Abstract:

The invention provides isolated nucleic acid and amino acid sequences of
Slo2 and Slo4, members of the Slo family of potassium channel proteins,
also known as "maxi" or BK potassium channel proteins. Also provided
herein are antibodies to Slo2 and Slo 4, methods of detecting Slo2 and
Slo 4, methods of screening for potassium channel activators and
inhibitors using biologically active Slo2 and Slo 4, and kits for
screening for activators and inhibitors of voltage-gated potassium
channels comprising Slo2 and Slo 4.

Claims:

1-46. (canceled)

47. An isolated Slo2 polypeptide comprising an alpha subunit of a Slo
potassium channel, the polypeptide:(i) forming, with at least one
additional Slo alpha subunit, a Slo potassium channel comprising the
characteristic of voltage-gating; and(ii) comprising a sequence having an
amino acid sequence of having at least 95% sequence identity to SEQ ID
NO:2.

48. The polypeptide of claim 47, wherein the potassium channel further
comprises the characteristic of rapid activation.

49. The polypeptide of claim 47, wherein the polypeptide has a molecular
weight of between about 134 kD to about 144 kD.

50. The polypeptide of claim 47, wherein the polypeptide comprises an
alpha subunit of a homomeric potassium channel.

51. The polypeptide of claim 47, wherein the polypeptide comprises an
alpha subunit of a heteromeric potassium channel.

Description:

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT

[0002]Not applicable.

BACKGROUND OF THE INVENTION

[0003]Potassium channels are involved in a number of physiological
processes, including regulation of heartbeat, dilation of arteries,
release of insulin, excitability of nerve cells, and regulation of renal
electrolyte transport. Potassium channels are thus found in a wide
variety of animal cells such as nervous, muscular, glandular, immune,
reproductive, and epithelial tissue. These channels allow the flow of
potassium in and/or out of the cell under certain conditions. For
example, the outward flow of potassium ions upon opening of these
channels makes the interior of the cell more negative, counteracting
depolarizing voltages applied to the cell. These channels are regulated,
e.g., by calcium sensitivity, voltage-gating, second messengers,
extracellular ligands, and ATP-sensitivity.

[0008]The present invention therefore provides, for the first time, a gene
encoding Slo 4, a new member of the Slo family and the Slo2/4 subfamily
of potassium channels. In addition, the present invention presents for
the first time the gene encoding human Slo2.

[0009]In one aspect, the present invention provides an isolated nucleic
acid encoding a Slo4 polypeptide comprising an alpha subunit of a Slo
potassium channel, the polypeptide: (i) forming, with at least one
additional Slo alpha subunit, a Slo potassium channel comprising the
characteristic of voltage-gating; and (ii) comprising a sequence having
at least 60% amino acid sequence identity to an amino acid sequence of
SEQ ID NO:4.

[0011]In one embodiment, the nucleic acid encodes an amino acid sequence
of SEQ ID NO:4. In another embodiment, the nucleic acid comprises a
nucleotide sequence of SEQ ID NO:3. In another embodiment, the nucleic
acid selectively hybridizes under moderately stringent hybridization
conditions to a nucleotide sequence of SEQ ID NO:3.

[0012]In one embodiment, the nucleic acid is amplified by at least one
pair of primers that selectively hybridize under stringent hybridization
conditions to the same sequence as the primers selected from the group
consisting of:

[0015]In another aspect, the present invention provides an isolated Slo4
polypeptide comprising an alpha subunit of a Slo potassium channel, the
polypeptide: (i) forming, with at least one additional Slo alpha subunit,
a Slo potassium channel comprising the characteristic of voltage-gating;
and (ii) comprising a sequence having at least 60% amino acid sequence
identity to an amino acid sequence of SEQ ID NO:4.

[0016]In another embodiment, the potassium channel further comprises the
characteristic of rapid activation.

[0017]In one embodiment, the polypeptide specifically binds to antibodies
generated against SEQ ID NO:4. In another embodiment, the polypeptide
comprises an alpha subunit of a homomeric or a heteromeric potassium
channel. In one embodiment, the polypeptide has a molecular weight of
between about 134 kD to about 144 kD.

[0018]In another embodiment, the polypeptide encodes human Slo4. In
another embodiment, the polypeptide has an amino acid sequence of SEQ ID
NO:4.

[0019]In one aspect, the invention provides an antibody that specifically
binds to the Slo4 polypeptide of the invention.

[0020]In another aspect, the present invention provides a method for
identifying a compound that increases or decreases ion flux through a
potassium channel, the method comprising the steps of: (i) contacting the
compound with a Slo4 polypeptide, the polypeptide (a) forming, with at
least one additional Slo alpha subunit, a Slo potassium channel having
the characteristic of voltage-gating; and (b) comprising a sequence
having at least 60% amino acid sequence identity to an amino acid
sequence of SEQ ID NO:4; and (ii) determining the functional effect of
the compound upon the potassium channel.

[0021]In one embodiment, the functional effect is a physical effect or a
chemical effect. In another embodiment, the functional effect is
determined by measuring ion flux, changes in ion concentrations, changes
in current or changes in voltage. In another embodiment, the functional
effect is determined by measuring ligand binding to the channel.

[0022]In one embodiment, the polypeptide is expressed in a eukaryotic host
cell or cell membrane. In another embodiment, the polypeptide is
recombinant.

[0023]In one aspect, the present invention provides a method for
identifying a compound that increases or decreases ion flux through a
potassium channel comprising a Slo4 polypeptide, the method comprising
the steps of: (i) entering into a computer system an amino acid sequence
of at least 25 amino acids of a Slo4 polypeptide or at least 75
nucleotides of a nucleic acid encoding the Slo4 polypeptide, the Slo4
polypeptide comprising a subsequence having at least 60% amino acid
sequence identity to an amino acid sequence of SEQ ID NO:4; (ii)
generating a three-dimensional structure of the polypeptide encoded by
the amino acid sequence; (iii) generating a three-dimensional structure
of the potassium channel comprising the Slo4 polypeptide; (iv) generating
a three-dimensional structure of the compound; and (v) comparing the
three-dimensional structures of the polypeptide and the compound to
determine whether or not the compound binds to the polypeptide.

[0024]In another aspect, the present invention provides a method of
modulating ion flux through a Slo potassium channel comprising a Slo4
polypeptide to treat disease in a subject, the method comprising the step
of administering to the subject a therapeutically effective amount of a
compound identified using the methods of the invention.

[0025]In another aspect, the present invention provides a method of
detecting the presence of hSlo4 in human tissue, the method comprising
the steps of: (i) isolating a biological sample; (ii) contacting the
biological sample with an hSlo4-specific reagent that selectively
associates with hSlo4; and, (iii) detecting the level of hSlo4-specific
reagent that selectively associates with the sample.

[0026]In one embodiment, the hSlo4-specific reagent is selected from the
group consisting of: hSlo4-specific antibodies, hSlo4-specific
oligonucleotide primers, and hSlo4-nucleic acid probes.

[0027]In another aspect, the present invention provides, in a computer
system, a method of screening for mutations of a human Slo4 gene, the
method comprising the steps of: (i) entering into the computer a first
nucleic acid sequence encoding a Slo4 polypeptide having an amino acid
sequence of SEQ ID NO:4, and conservatively modified versions thereof;
(ii) comparing the first nucleic acid sequence with a second nucleic acid
sequence having substantial identity to the first nucleic acid sequence;
and (iii) identifying nucleotide differences between the first and second
nucleic acid sequences.

[0028]In one embodiment, the second nucleic acid sequence is associated
with a disease state.

[0029]In one aspect, the present invention provides an isolated nucleic
acid encoding a Slo2 polypeptide comprising an alpha subunit of a Slo
potassium channel, the polypeptide: (i) forming, with at least one
additional Slo alpha subunit, a Slo potassium channel comprising the
characteristic of voltage-gating; and (ii) comprising an amino acid
sequence of SEQ ID NO:2.

[0030]In another aspect, the present invention provides an isolated
nucleic acid encoding a Slo2 polypeptide, wherein the nucleic acid is
amplified by primers that selectively hybridize under stringent
hybridization conditions to the same sequence as the primers selected
from the group consisting of:

[0034]In one aspect, the present invention provides an isolated Slo2
polypeptide comprising an alpha subunit of a Slo potassium channel, the
polypeptide: (i) forming, with at least one additional Slo alpha subunit,
a Slo potassium channel comprising the characteristic of voltage-gating;
and (ii) comprising a sequence having an amino acid sequence of SEQ ID
NO:2.

[0035]In one embodiment, the potassium channel further comprises the
characteristic of rapid activation.

[0036]In one embodiment, the polypeptide encoded by the nucleic acid
comprises an alpha subunit of a heteromeric or homomeric potassium
channel. In another embodiment, the polypeptide has a molecular weight of
between about 134 kD to about 144 kD.

[0037]In one aspect, present the invention provides an antibody that
specifically binds to the Slo4 polypeptide of the invention.

[0038]In another aspect, the present invention provides a method for
identifying a compound that increases or decreases ion flux through a
potassium channel, the method comprising the steps of: (i) contacting the
compound with a Slo4 polypeptide, the polypeptide (a) forming, with at
least one additional Slo alpha subunit, a Slo potassium channel having
the characteristic of voltage-gating; and (b) comprising a sequence
having an amino acid sequence of SEQ ID NO:2; and (ii) determining the
functional effect of the compound upon the potassium channel.

[0039]In one embodiment, the functional effect is a physical effect or a
chemical effect. In another embodiment, the functional effect is
determined by measuring ion flux, changes in ion concentrations, changes
in current or changes in voltage. In another embodiment, the functional
effect is determined by measuring ligand binding to the channel.

[0040]In one embodiment, the polypeptide is expressed in a eukaryotic host
cell or cell membrane. In another embodiment, the polypeptide is
recombinant.

[0041]In another aspect, the present invention provides a method for
identifying a compound that increases or decreases ion flux through a
potassium channel comprising a Slo2 polypeptide, the method comprising
the steps of: (i) entering into a computer system an amino acid sequence
of SEQ ID NO:2; (ii) generating a three-dimensional structure of the
polypeptide encoded by the amino acid sequence; (iii) generating a
three-dimensional structure of the potassium channel comprising the Slo2
polypeptide; (iv) generating a three-dimensional structure of the
compound; and (v) comparing the three-dimensional structures of the
polypeptide and the compound to determine whether or not the compound
binds to the polypeptide.

[0042]In another aspect, the present invention provides a method of
modulating ion flux through a Slo potassium channel comprising a Slo2
polypeptide to treat disease in a subject, the method comprising the step
of administering to the subject a therapeutically effective amount of a
compound identified using the methods of the invention.

[0043]In another aspect, the present invention provides a method of
detecting the presence of hSlo2 in human tissue, the method comprising
the steps of: (i) isolating a biological sample; (ii) contacting the
biological sample with an hSlo2-specific reagent that selectively
associates with hSlo2; and, (iii) detecting the level of hSlo2-specific
reagent that selectively associates with the sample.

[0044]In one embodiment, the hSlo2-specific reagent is selected from the
group consisting of: hSlo2-specific antibodies, hSlo2-specific
oligonucleotide primers, and hSlo2-nucleic acid probes.

[0045]In another aspect, the present invention provides, in a computer
system, a method of screening for mutations of a human Slo2 gene, the
method comprising the steps of: (i) entering into the computer a first
nucleic acid sequence encoding a Slo2 polypeptide having an amino acid
sequence of SEQ ID NO:2, and conservatively modified versions thereof;
(ii) comparing the first nucleic acid sequence with a second nucleic acid
sequence having substantial identity to the first nucleic acid sequence;
and (iii) identifying nucleotide differences between the first and second
nucleic acid sequences.

[0046]In one embodiment, the second nucleic acid sequence is associated
with a disease state.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1. Amino acid alignment of the complete human Slo2 amino acid
sequence to the amino acids sequences of rat SLACK and the partial human
cDNA KIAA1422. Identical residues are shaded and amino acid numbers are
given at the left margin. Human Slo2 and rat SLACK are 93% identical and
are probably orthologous genes. The pattern of divergence between human
Slo2 and rat SLACK is not typical for orthologous potassium channels.
KIAA1422 is a partial cDNA from the hSlo2 gene, but differs in several
key ways. First, it has an alternative amino terminus that is highly
divergent from those of human Slo2 and rat SLACK. It is also clearly
truncated on the 3' end. The human Slo2 DNA and amino acid sequences can
not be readily predicted from KIAA1422 or rat SLACK.

[0048]FIG. 2. Amino acid alignment of human Slo2 and human Slo4. Identical
residues are shaded and amino acid numbers are given at the left margin.
Six predicted transmembrane domains are underlined with solid lines. The
potassium channel "signature sequence" or pore loop is underlined with a
dotted line. A dashed line indicates a tail region that has been
implicated in Slo channel calcium and chloride gating (Yuan et al., Nat.
Neurosci. 3:771-779 (2000); Wei et al., Neuron, 13:671-681 (1994)). The
two amino acid sequences are approximately 70% identical.

[0049]FIG. 3. Phylogenetic tree of the Slo potassium channel gene family.
Two main branches define two distinct gene subfamilies. Human Slo2 and
Slo4 are in a subfamily that also includes rat SLACK and the C. elegans
Slo2 gene (ceSlo2). Note that C. elegans Slo2 branches separately from
both Slo2 and Slo4, suggesting that it is not orthologous to either of
these genes.

[0050]FIG. 4. Messenger RNA expression patterns of human Slo2. Blots were
probed with a 32P-labeled Slo2 PCR fragment and hybridized with
standard high stringency procedures. (A) Northern blot of Slo2. Strong
bands of approximately 5 Kb are seen in brain and skeletal muscle. Weaker
bands of 6+Kb are also present and may represent alternative splice
variants or incompletely processed RNAs. Similar bands are also seen in
heart and spleen. (B) mRNA dot blot hybridized with the same Slo2 probe.
Note the strong signals seen in CNS tissues, skeletal muscle, spleen,
heart, pituitary and ovary.

[0051]FIG. 5. Functional expression of human Slo2 in Xenopus oocytes. (A)
Slo2 currents elicited by depolarizing voltage steps from a holding
potential of -120 mV up to +40 mV. Steps were taken in 20 mV increments
and tail currents were measured at -60 mV. Note that Slo2 current is
clearly seen at voltages as low as -80 mV. Activation at -100 mV may be
obscured because this in near the potassium equilibrium potential. (B)
Currents elicited from an oocyte expressing Slo2 with 3 second voltage
ramps ranging from -160 mV to +80 mV. Ramps were run with either 2 mM
external potassium (1) or 50 mM external potassium (2). Note the large
positive shift in reversal potential of the current at 50 mM potassium
(right arrow). Also note the large inward current in 50 mM potassium that
is seen at the resting voltage of -90 mV (left arrow). This indicates
significant activation of Slo2 at -90 mV. It is unmasked by 50 mM
potassium which shifts the reversal potential away from -90 mV, providing
a large driving force for potassium entry.

[0052]FIG. 6. Functional expression of human Slo4 in Xenopus oocytes. (A)
Slo4 currents elicited by depolarizing voltage steps from -80 mV up to
+80 mV. Steps were taken in 20 mV increments from a holding potential of
-100 mV, and tail currents were measured at -60 mV. The Slo4 current is
clearly seen at -60 mV, and some Slo4 current is present even at -80 mV.
(B) Currents elicited from an oocyte expressing Slo4 with 3 second
voltage ramps ranging from -160 mV to +80 mV. Ramps were run with either
2 mM external potassium (1) or 20 mM external potassium (2). Note the
large positive shift in reversal potential of the current in 20 mM
potassium (right arrow). Also note that some inward current in 20 mM
potassium is seen at the resting voltage of -90 mV (left arrow). This
indicates activation of Slo4 at -90 mV.

[0053]FIG. 7. (A) Human northern blot hybridized with a 32P-labeled
Slo4 cDNA probe. Marks at the left margin indicate molecular weight in
kilobases (Kb). Lane numbers are given at the top. The following is a
list of the tissues in these lanes: 1) whole brain, 2) heart, 3) skeletal
muscle, 4) colon, 5) thymus, 6) spleen, 7) kidney, 8) liver, 9) small
intestine, 10) placenta, 11) lung, 12) peripheral blood leukocytes. A
transcript of approximately 5.5 Kb is labeled in most of these tissues.
Expression is highest in the liver, with high level expression also being
found in the brain and heart. Lower levels of expression are detected in
skeletal muscle, colon, spleen, kidney, small intestine, placenta and
lung. Larger transcripts of approximately 9 Kb and 13 Kb are seen in
brain and heart. These may represent alternative transcripts or
incompletely processed transcripts. A 4.5 Kb transcript is seen in lung,
and may represent an alternative transcript; it is long enough to encode
a complete Slo4 protein. (B) mRNA dot blot hybridized with the same probe
as in (A). The highest levels of expression are seen in liver, fetal
brain, fetal kidney and fetal liver. High levels of expression are also
seen in testis, fetal lung, most brain regions, the atrium, the GI track,
lung, placenta and bladder. Expression is detectable in many other
tissues on the blot. Based on comparison with the northern results in
(A), these low signals are likely to represent Slo4 expression and not
background non-specific hybridization.

DETAILED DESCRIPTION OF THE INVENTION

[0054]The present invention provides for the first time nucleic acids
encoding Slo4 potassium channels. The present invention also provides the
sequence of human Slo2. These polypeptide monomers are members of the Slo
family of potassium channels, and the Slo2/4 subfamily. Members of this
family are polypeptide subunits of potassium channels having six
transmembrane regions and a pore-loop domain, as well as a cytoplasmic
tail.

[0055]Both human Slo2 and Slo4 are expressed in the heart and central
nervous system and appear to contribute to the modulation of neuronal
excitability, because both Slo2 and Slo4 begin to activate in a voltage
range below the typical thresholds for action potential generation. They
also appear to be involved in action potential repolarization and
refractory period and the control of neurotransmitter release, as is the
case for other Slo family members. The expression of human Slo2 and Slo4
in peripheral tissues such as skeletal muscle, heart and spleen indicate
that they may also be involved in regulating muscle contraction, heart
rate, airway tone, inflammation, and lymphocyte proliferation. In
addition, modulators of Slo2 and Slo4 should be useful in treating
disorders of neuronal excitability related to increased levels of
neuronal activity or abnormal neurotransmitter release. This includes
neuropathic pain, epilepsy and seizure disorders, depression and other
psychotic disorders such as bipolar disease and schizophrenia, migraine
and anxiety. Modulators could also be useful in treating disorders of
learning and memory caused by diseases such as Alzheimer's, or to enhance
learning and memory in the aging population. In some cells, enhancement
of Slo2 and Slo4 currents will cause greater hyperpolarization and
decrease depolarization-based calcium influxes, providing
neuroprotection. In other cells in which the calcium influx is
independent of voltage, blockers of Slo2 and Slo4 may reduce the driving
force for calcium entry, again providing neuroprotection.

[0056]The invention therefore provides methods of screening for activators
and inhibitors of potassium channels that contain a Slo2 or a Slo4 alpha
subunit. Such modulators of potassium channel activity are useful for
treating disorders, including CNS disorders, such as neuropathic pain,
epilepsy and other seizure disorders, migraines, anxiety, psychotic
disorders such as schizophrenia, bipolar disease, and depression. Such
modulators are also useful as neuroprotective agents (e.g., to prevent
stroke). Modulators could also be useful in treating cognitive disorders
of learning and memory caused by diseases such as Alzheimer's, or to
enhance learning and memory in the aging population, as well providing
neuroprotection. Finally, such modulators could be useful for treating
hypercontractility of muscles, cardiac arrhythmias, inflammation, asthma,
and as immunosuppressants or stimulants.

[0057]Furthermore, the invention provides assays for Slo2 and Slo4
activity where Slo2 or Slo4 acts as a direct or indirect reporter
molecule. Such uses of Slo2 and Slo4 as a reporter molecule in assay and
detection systems have broad applications, e.g., Slo2 or Slo4 can be used
as a reporter molecule to measure changes in potassium concentration,
membrane potential, current flow, ion flux, transcription, signal
transduction, receptor-ligand interactions, second messenger
concentrations, in vitro, in vivo, and ex vivo. In one embodiment, Slo2
or Slo4 can be used as an indicator of current flow in a particular
direction (e.g., outward or inward potassium flow), and in another
embodiment, Slo2 or Slo4 can be used as an indirect reporter via
attachment to a second reporter molecule such as green fluorescent
protein.

[0058]The invention also provides for methods of detecting Slo2 and Slo4
nucleic acid and protein expression, allowing investigation of the
channel diversity provided by Slo2 and Slo4 family members, as well as
diagnosis of disorders, including CNS disorders, such as neuropathic
pain, epilepsy and other seizure disorders, migraines, anxiety, psychotic
disorders such as schizophrenia, bipolar disease, and depression,
cognitive disorders of learning and memory caused by diseases such as
Alzheimer's, hypercontractility of muscles, cardiac arrhythmias,
inflammation, and asthma.

[0059]Finally, the invention provides for a method of screening for
mutations of Slo2 and Slo4 genes or proteins. The invention includes, but
is not limited to, methods of screening for mutations in Slo2 or Slo4
with the use of a computer. Similarly, the invention provides for methods
of identifying the three-dimensional structure of Slo2 and Slo4
polypeptides, e.g., human Slo2 and human Slo4, as well as the resulting
computer readable images or data that comprise the three dimensional
structure of Slo2 and Slo4 polypeptides. Other methods for screening for
mutations of Slo2 and Slo4 genes or proteins include high density
oligonucleotide arrays, PCR, immunoassays and the like.

[0060]Functionally, Slo2 and Slo4 polypeptides are alpha subunits of a Slo
potassium channel. Slo2 and Slo4 potassium channels are potassium
selective and voltage gated (e.g., the number of channels that open
during a voltage step increases with increasing depolarization) In
addition, these channels may be regulated by other means, e.g., calcium,
chloride, or pH (see, e.g., Yuan et al., Nat. Neurosci. 8:771-779 (2000);
see also Schreiber, supra, and Butler, supra). Typically, such channels
are heteromeric or homomeric and contain four alpha subunits or monomers
each with six or seven transmembrane domains. Heteromeric Slo channels
can comprise one or more Slo2 or Slo4 alpha subunits along with one or
more additional alpha subunits from the Slo family (see, e.g., McManus et
al., Neuron 14:645-650 (1995); Schopperle et al., Neuron 20:565-573
(1998); Brenner et al., J. Biol. Chem. 275:6453-6461 (1999); and WO
0050444). Slo2 and Slo4 channels may also be homomeric. In addition, such
homomeric channels may comprise one or more auxiliary beta subunits. The
presence of Slo2 or Slo4 in a potassium channel may also modulate the
activity of the heteromeric channel and thus enhance channel diversity,
for example by altering a channel characteristic such as conductance.
Channel diversity is also enhanced with alternatively spliced forms of
Slo2 and Slo4 genes.

[0062]Structurally, the nucleotide sequence of human Slo2 (SEQ ID NO:1)
encodes a polypeptide monomer of about 1235 amino acids (SEQ ID NO:2)
with a predicted molecular weight of about 139 Kd, and a range of
approximately 134 Kd to 164 Kd.

[0063]The present invention also provides polymorphic variants of the
human Slo2 depicted in SEQ ID NO:2: variant #1, in which a serine residue
is substituted for the alanine residue at amino acid position 24; variant
#2, in which a threonine residue is substituted for the alanine residue
at amino acid position 39; variant #3, in which a threonine residue is
substituted for the serine residue at amino acid position 113; and
variant #4, in which a lysine residue is substituted for the arginine
residue at amino acid position 1105.

[0064]Structurally, the nucleotide sequence of human Slo4 (SEQ ID NO:3)
encodes a protein of about 1135 amino acids (SEQ ID NO:4) with a
predicted molecular weight of about 130 Kd, and a range of approximately
125 Kd to 135 Kd.

[0065]The present invention also provides polymorphic variants of the
human Slo4 depicted in SEQ ID NO:4: variant #1, in which a valine residue
is substituted for the methionine residue at amino acid position 39;
variant #2, in which a alanine residue is substituted for the serine
residue at amino acid position 87; variant #3, in which a serine residue
is substituted for the threonine residue at amino acid position 547; and
variant #4, in which an isoleucine residue is substituted for the valine
residue at amino acid position 1098.

[0066]Specific regions of Slo2 or Slo4 nucleotide and amino acid sequence
may be used to identify Slo2 or Slo4 polymorphic variants, interspecies
homologs, and alleles. This identification can be made in vitro, e.g.,
under stringent hybridization conditions and sequencing, or by using the
sequence information in a computer system for comparison with other
nucleotide sequences, or using antibodies raised to Slo2 or Slo4.
Typically, identification of Slo2 or Slo4 polymorphic variants,
orthologs, and alleles is made by comparing the amino acid sequence (or
the nucleic acid encoding the amino acid sequence) to SEQ ID NO:2 or 4,
or a conserved region such as the core transmembrane domain (pore loop
and S1-S6 transmembrane domains), or the tail domain. Amino acid identity
of approximately at least 60% or above, preferably 70%, 65%, 75%, 80%,
85%, most preferably 90-95% or above in the full length amino acid
sequence typically demonstrates that a protein is a Slo2 or Slo4
polymorphic variant, interspecies homolog, or allele. Amino acid identity
of approximately at least 60%, 65%, 70%, or 75%, 75% or above, preferably
80%, 85%, most preferably 90-95% or above in the core transmembrane
domain (S1-S6 plus the pore loop) or the C-terminal cytoplasmic tail
domain typically demonstrates that a protein is a Slo2 or Slo4
polymorphic variant, interspecies homolog, or allele. Sequence comparison
is typically performed using the BLAST or BLAST 2.0 algorithm with
default parameters, discussed below.

[0067]Slo2 or Slo4 polymorphic variants, interspecies homologs, and
alleles can be confirmed by expressing or co-expressing the putative Slo2
or Slo4 polypeptide monomer and examining whether it forms a potassium
channel with Slo family functional characteristics, such as voltage
gating, and Slo2 or Slo4 characteristics such as relatively rapid
activation and deactivation. This assay is used to demonstrate that a
protein having about 60% or greater, preferably 65%, 70%, 75%, 80%, 85%,
90%, or 95% or greater amino acid identity to a conserved region of Slo2
or Slo4 shares the same functional characteristics as Slo2 or Slo4 and is
therefore a species of Slo2 or Slo4. Typically, human Slo2 or Slo4 having
the amino acid sequence of SEQ ID NO:2 or 4 is used as a positive control
in comparison to the putative Slo2 or Slo4 protein to demonstrate the
identification of a Slo2 or Slo4 polymorphic variant, ortholog,
conservatively-modified variant, mutant, or allele.

[0068]Slo2 or Slo4 nucleotide and amino acid sequence information may also
be used to construct models of Slo voltage-gated potassium channels in a
computer system. These models are subsequently used to identify compounds
that can activate or inhibit voltage-gated potassium channels comprising
Slo2 or Slo4 polypeptides. Such compounds that modulate the activity of
channels comprising Slo2 or Slo4 polypeptides can be used to investigate
the role of Slo2 or Slo4 polypeptides in modulation of channel activity
and in channel diversity.

[0069]The isolation of biologically active human Slo2 and human Slo4 for
the first time provides a means for assaying for inhibitors and
activators of voltage-gated potassium channels that comprise Slo2 or Slo4
subunits. Biologically active Slo2 or Slo4 polypeptides are useful for
testing inhibitors and activators of voltage-gated potassium channels
comprising subunits of Slo2 or Slo4 and/or other Slo members such as Slo1
or Slo3, using in vivo and in vitro expression that measure, e.g.,
changes in voltage or current. Such activators and inhibitors identified
using a potassium channel comprising at least one Slo2 or Slo4 subunit,
optionally up to four Slo2 or Slo4 subunits, can be used to further study
voltage gating, channel kinetics and conductance properties of potassium
channels. Such activators and inhibitors are useful as pharmaceutical
agents for treating diseases involving abnormal ion flux, e.g., CNS
disorders, such as neuropathic pain, epilepsy and other seizure
disorders, migraines, anxiety, psychotic disorders such as schizophrenia,
bipolar disease, and depression. Such modulators are also useful as
neuroprotective agents (e.g., to prevent stroke). Modulators could also
be useful in treating cognitive disorders of learning and memory caused
by diseases such as Alzheimer's, or to enhance learning and memory in the
aging population, as well providing neuroprotection. Finally, such
modulators could be useful for treating hypercontractility of muscles,
cardiac arrhythmias, inflammation, asthma, and as immunosuppressants or
stimulants.

[0070]Methods of detecting Slo2 or Slo4 nucleic acids and polypeptides and
expression of channels comprising Slo2 or Slo4 polypeptides are also
useful for diagnostic applications for diseases involving abnormal ion
flux, e.g., as described above. For example, chromosome localization of
the gene encoding human Slo2 or Slo4 can be used to identify diseases
caused by and associated with Slo2 or Slo4. Methods of detecting Slo2 or
Slo4 are also useful for examining the role of Slo2 or Slo4 in channel
diversity and modulation of channel activity.

II. DEFINITIONS

[0071]As used herein, the following terms have the meanings ascribed to
them unless specified otherwise.

[0072]"Slo4" refers to a polypeptide that is a subunit or monomer of a Slo
or Slo4 potassium channel, and a member of the Slo family. When Slo4 is
part of a homomeric potassium channel, the channel has the characteristic
of voltage gating and rapid deactivation. In addition, when Slo4 is part
of a heteromeric potassium channel, it can confer altered
characteristics. Slo4 has a core transmembrane domain corresponding to
amino acids 64-282 of SEQ ID NO:4, which comprises the pore loop domain
and the S1-S6 transmembrane domains. Slo4 also has a C-terminal
cytoplasmic tail domain from amino acids 336-1135 of SEQ ID NO:4.

[0073]The term Slo4 therefore refers to Slo4 polymorphic variants,
alleles, mutants, and orthologs (interspecies homologs) that: (1) have a
sequence that has greater than about 60% amino acid sequence identity,
preferably about 65%, 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence
identity using a sequence comparison algorithm such as BLASTP with the
parameters described herein, to a Slo4 amino acid sequence of SEQ ID NO:4
or a conserved region such as the core transmembrane domain or the
C-terminal cytoplasmic tail; (2) bind to antibodies, e.g., polyclonal or
monoclonal antibodies, raised against an immunogen comprising an amino
acid sequence of SEQ ID NO:4 or an immunogenic fragment thereof, and
conservatively modified variants thereof; (3) specifically hybridize
under highly and/or moderately stringent hybridization conditions to a
sequence of SEQ ID NO:3, and conservatively modified variants thereof; or
(4) are amplified by primers that specifically hybridize under highly
and/or moderately stringent hybridization conditions to the same sequence
as a primer set selected from the group consisting of SEQ ID NOS:23-31.

[0074]"Slo2" refers to a polypeptide that is a subunit or monomer of a Slo
or Slo4 potassium channel, and a member of the Slo family. When Slo2 is
part of a homomeric potassium channel, the channel has the characteristic
of voltage gating and rapid deactivation. In addition, when Slo2 is part
of a heteromeric potassium channel, it can confer altered
characteristics. Slo2 has a core transmembrane domain from amino acids
98-335 of SEQ ID NO:2, which comprises the pore loop domain and the S1-S6
transmembrane domains. Slo2 also has a C-terminal cytoplasmic tail domain
from amino acids 283-1235 of SEQ ID NO:2.

[0075]The term Slo2 therefore refers to Slo4 polymorphic variants,
alleles, and mutants that: (1) have amino acid sequence identity greater
than about 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity
using a sequence comparison algorithm such as BLASTP with the parameters
described herein, to a Slo2 amino acid sequence of SEQ ID NO:2 or a
conserved region such as the core transmembrane domain or the C-terminal
cytoplasmic tail; (2) bind to antibodies, e.g., polyclonal or monoclonal
antibodies, raised against an immunogen comprising an amino acid sequence
of SEQ ID NO:2 or an immunogenic fragment thereof, and conservatively
modified variants thereof; (3) specifically hybridize under highly and/or
moderately stringent hybridization conditions to a sequence of SEQ ID
NO:1, and conservatively modified variants thereof; or (4) are amplified
by primers that specifically hybridize under highly and/or moderately
stringent hybridization conditions to the same sequence as a primer set
selected from the group consisting of SEQ ID NOS:5-22, in particular a
primer set with at least one primer selected from the group consisting of
SEQ ID NOS:18-21.

[0076]The phrase "voltage-gated" activity or "voltage-gating" refers to a
characteristic of a potassium channel composed of individual polypeptide
monomers or subunits. Generally, the probability of a voltage-gated
potassium channel opening increases as a cell is depolarized.
Voltage-gated potassium channels primarily allow efflux of potassium
because they have greater probabilities of being open at membrane
potentials more positive than the equilibrium potential for potassium
(EK) in typical cells. EK, or the equilibrium potential for
potassium, depends on the relative concentrations of potassium found
inside and outside the cell membrane, and is typically between -60 and
-100 mV for mammalian cells. EK is the membrane potential at which
there is no net flow of potassium ion because the electrical potential
(i.e., voltage potential) driving potassium influx is balanced by the
concentration gradient (the [K.sup.+] potential) directing potassium
efflux. This value is also known as the "reversal potential" or the
"Nernst" potential for potassium. Some voltage-gated potassium channels
undergo inactivation, which can reduce potassium efflux at higher
membrane potentials. Potassium channels can also allow potassium influx
in certain instances when they remain open at membrane potentials
negative to EK (see, e.g., Adams & Nonner, in Potassium Channels,
pp. 40-60 (Cook, ed., 1990)). The characteristic of voltage gating can be
measured by a variety of techniques for measuring changes in current flow
and ion flux through a channel, e.g., by changing the [K.sup.+] of the
external solution and measuring the activation potential of the channel
current (see, e.g., U.S. Pat. No. 5,670,335), by measuring current with
patch clamp techniques or voltage clamp under different conditions, and
by measuring ion flux with radiolabeled tracers or voltage-sensitive dyes
under different conditions.

[0077]"Large conductance" refers to the conductance of certain potassium
channels. In native cells, conductances of these channels range from
about 40 to 50 pS to over 200 pS (see, e.g., Latorre et al., Annu. Rev.
Physio. 51:385-399 (19891)). For example, Slo1 and Slo3 channels have
conductances near the upper end of this range, while Slo2 channels have
conductances close to the lower end.

[0078]"Homomeric channel" refers to a Slo2 or a Slo4 channel composed of
identical alpha subunits, whereas "heteromeric channel" refers to a Slo
channel composed of at least one Slo2 or Slo4 alpha subunit, plus at
least one other different type of alpha subunit from another Slo family
member such as Slo1 or Slo3. Both homomeric and heteromeric channels can
include auxiliary beta subunits. Typically, the channel is composed of
four alpha subunits and the channel can be heteromeric or homomeric.

[0079]A "beta subunit" is a polypeptide monomer that is an auxiliary
subunit of a potassium channel composed of alpha subunits; however, beta
subunits alone cannot form a channel (see, e.g., U.S. Pat. No.
5,776,734). Beta subunits are known, for example, to increase the number
of channels by helping the alpha subunits reach the cell surface, change
activation kinetics, and change the sensitivity of natural ligands
binding to the channels. Beta subunits can be outside of the pore region
and associated with alpha subunits comprising the pore region. They can
also contribute to the external mouth of the pore region.

[0080]The phrase "functional effects" in the context of assays for testing
compounds affecting a channel comprising Slo2 or Slo4 includes the
determination of any parameter that is indirectly or directly under the
influence of the channel. It includes e.g., direct, physical effects,
such as ligand binding, and indirect, chemical or phenotypic effects,
e.g., changes in ion flux and membrane potential, and other physiologic
effects such as increases or decreases of transcription or hormone
release. "Functional effects" include in vitro (biochemical or ligand
binding assays using, e.g., isolated protein, cell lysates or cell
membranes), in vivo (cell- and animal-based assays), and ex vivo
activities.

[0081]"Determining the functional effect" refers to examining the effect
of a compound that has a direct physical effect on a Slo2 or Slo4 subunit
or channel comprising a Slo2 or a Slo4 subunit, e.g., ligand binding, or
indirect chemical or phenotypic effects on channel comprising a Slo2 or a
Slo4 subunit, e.g., increases or decreases ion flux in a cell or cell
membrane. The ion flux can be any ion that passes through a channel and
analogues thereof, e.g., potassium, rubidium. Preferably, the term refers
to the functional effect of the compound on the channels comprising Slo2
or Slo4, e.g., changes in ion flux including radioisotopes, current
amplitude, membrane potential, current flow, conductance, transcription,
protein binding, phosphorylation, dephosphorylation, second messenger
concentrations (cAMP, cGMP, Ca2+, IP3), ligand binding, changes
in ion concentration, and other physiological effects such as hormone and
neurotransmitter release, as well as changes in voltage and current. Such
functional effects can be measured by any means known to those skilled in
the art, e.g., patch clamping, voltage-sensitive dyes, ion sensitive
dyes, whole cell currents, radioisotope efflux, inducible markers, and
the like.

[0082]"Inhibitors," "activators" or "modulators" of voltage-gated
potassium channels comprising a Slo2 or a Slo4 polypeptide refer to
inhibitory or activating molecules identified using in vitro and in vivo
assays for Slo2 or a Slo4 channel function. Inhibitors are compounds that
decrease, block, prevent, delay activation, inactivate, desensitize, or
down regulate the channel, e.g., antagonists. Activators are compounds
that increase, open, activate, facilitate, enhance activation, sensitize
or up regulate channel activity, e.g., agonists. Such assays for
inhibitors and activators include e.g., expressing a Slo2 or a Slo4
polypeptide in cells, cell extracts, or cell membranes and then measuring
flux of ions through the channel and determining changes in polarization
(i.e., electrical potential). Alternatively, cells expressing endogenous
Slo2 or a Slo4 channels can be used in such assays. Isolated naturally
occurring or recombinant Slo2 or Slo4-comprising channels or Slo2 or Slo4
subunits, or cell extracts containing the same can also be used in ligand
binding assays to identify such modulators. To examine the extent of
inhibition, samples or assays comprising a Slo2 or a Slo4 subunit or
channel are treated with a potential activator or inhibitor and are
compared to control samples without the inhibitor. Control samples
(untreated with inhibitors) are assigned a relative Slo2 or a Slo4
activity value of 100% Inhibition of channels comprising Slo2 or a Slo4
is achieved when the Slo2 or a Slo4 activity value relative to the
control is about 90%, preferably 50%, more preferably 25-0%. Activation
of channels comprising Slo2 or a Slo4 is achieved when the Slo2 or a Slo4
activity value relative to the control is 110%, more preferably 150%,
most preferably at least 200-500% higher or 1000% or higher.

[0083]"Biologically active" Slo2 or a Slo4 polypeptides refers to Slo2 or
a Slo4 polypeptides that have the ability to form a potassium channel
having the characteristic of voltage-gating and rapid deactivation,
tested as described above.

[0084]The terms "isolated," "purified," or "biologically pure" refer to
material that is substantially or essentially free from components that
normally accompany it as found in its native state. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high performance
liquid chromatography. A protein that is the predominant species present
in a preparation is substantially purified. In particular, an isolated
Slo2 or a Slo4 nucleic acid is separated from open reading frames that
flank the Slo2 or Slo4 gene and encode proteins other than Slo2 or Slo4.
The term "purified" denotes that a nucleic acid or protein gives rise to
essentially one band in an electrophoretic gel. Particularly, it means
that the nucleic acid or protein is at least 85% pure, more preferably at
least 95% pure, and most preferably at least 99% pure. Isolated or
purified Slo2 and 4 can be recombinant or naturally occurring.

[0085]The term "test compound" or "drug candidate" or "modulator" or
grammatical equivalents as used herein describes any molecule, either
naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from
about 5 to about 25 amino acids in length, preferably from about 10 to 20
or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids
in length), small organic molecule, polysaccharide, lipid (e.g., a
sphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., to be
tested for the capacity to directly or indirectly modulation lymphocyte
activation. The test compound can be in the form of a library of test
compounds, such as a combinatorial or randomized library that provides a
sufficient range of diversity. Test compounds are optionally linked to a
fusion partner, e.g., targeting compounds, rescue compounds, dimerization
compounds, stabilizing compounds, addressable compounds, and other
functional moieties. Conventionally, new chemical entities with useful
properties are generated by identifying a test compound (called a "lead
compound") with some desirable property or activity, e.g., inhibiting
activity, creating variants of the lead compound, and evaluating the
property and activity of those variant compounds. Often, high throughput
screening (HTS) methods are employed for such an analysis.

[0086]A "small organic molecule" refers to an organic molecule, either
naturally occurring or synthetic, that has a molecular weight of more
than about 50 daltons and less than about 5000 daltons, preferably less
than about 2000 daltons, preferably between about 100 to about 1000
daltons, more preferably between about 200 to about 500 daltons.

[0087]The term "pain" refers to all categories of pain, including pain
that is described in terms of stimulus or nerve response, e.g., somatic
pain (normal nerve response to a noxious stimulus) and neuropathic pain
(abnormal response of a injured or altered sensory pathway, often without
clear noxious input); pain that is categorized temporally, e.g., chronic
pain and acute pain; pain that is categorized in terms of its severity,
e.g., mild, moderate, or severe; and pain that is a symptom or a result
of a disease state or syndrome, e.g., inflammatory pain, cancer pain,
AIDS pain, arthropathy, migraine, trigeminal neuralgia, cardiac
ischaemia, and diabetic neuropathy (see, e.g., Harrison's Principles of
Internal Medicine, pp. 93-98 (Wilson et al., eds., 12th ed. 1991);
Williams et al., J. of Medicinal Chem. 42:1481-1485 (1999), herein each
incorporated by reference in their entirety).

[0088]"Somatic" pain, as described above, refers to a normal nerve
response to a noxious stimulus such as injury or illness, e.g., trauma,
burn, infection, inflammation, or disease process such as cancer, and
includes both cutaneous pain (e.g., skin, muscle or joint derived) and
visceral pain (e.g., organ derived).

[0089]"Neuropathic" pain, as described above, refers to pain resulting
from injury to or chronic changes in peripheral and/or central sensory
pathways, where the pain often occurs or persists without an obvious
noxious input.

[0090]"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. The term
encompasses nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally occurring,
and non-naturally occurring, which have similar binding properties as the
reference nucleic acid, and which are metabolized in a manner similar to
the reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic
acids (PNAs).

[0092]A particular nucleic acid sequence also implicitly encompasses
"splice variants." Similarly, a particular protein encoded by a nucleic
acid implicitly encompasses any protein encoded by a splice variant of
that nucleic acid. "Splice variants," as the name suggests, are products
of alternative splicing of a gene. After transcription, an initial
nucleic acid transcript may be spliced such that different (alternate)
nucleic acid splice products encode different polypeptides. Mechanisms
for the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same nucleic
acid by read-through transcription are also encompassed by this
definition. Any products of a splicing reaction, including recombinant
forms of the splice products, are included in this definition. An example
of potassium channel splice variants is discussed in Leicher, et al., J.
Biol. Chem. 273(52):35095-35101 (1998).

[0093]The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. The
terms apply to amino acid polymers in which one or more amino acid
residue is an artificial chemical mimetic of a corresponding naturally
occurring amino acid, as well as to naturally occurring amino acid
polymers and non-naturally occurring amino acid polymer.

[0094]The term "amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics that
function in a manner similar to the naturally occurring amino acids.
Naturally occurring amino acids are those encoded by the genetic code, as
well as those amino acids that are later modified, e.g., hydroxyproline,
γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers
to compounds that have the same basic chemical structure as a naturally
occurring amino acid, i.e., an α carbon that is bound to a
hydrogen, a carboxyl group, an amino group, and an R group, e.g.,
homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium. Such analogs have modified R groups (e.g., norleucine) or
modified peptide backbones, but retain the same basic chemical structure
as a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in a
manner similar to a naturally occurring amino acid.

[0095]Amino acids may be referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by the
IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may
be referred to by their commonly accepted single-letter codes.

[0096]"Conservatively modified variants" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic acids
which encode identical or essentially identical amino acid sequences, or
where the nucleic acid does not encode an amino acid sequence, to
essentially identical sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode any
given protein. For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded polypeptide.
Such nucleic acid variations are "silent variations," which are one
species of conservatively modified variations. Every nucleic acid
sequence herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize that
each codon in a nucleic acid (except AUG, which is ordinarily the only
codon for methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical molecule.
Accordingly, each silent variation of a nucleic acid which encodes a
polypeptide is implicit in each described sequence.

[0097]As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the encoded
sequence is a "conservatively modified variant" where the alteration
results in the substitution of an amino acid with a chemically similar
amino acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.

[0098]The following eight groups each contain amino acids that are
conservative substitutions for one another:

1) Alanine (A), Glycine (G);

[0099]2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

[0100](see, e.g., Creighton, Proteins (1984)).

[0101]Macromolecular structures such as polypeptide structures can be
described in terms of various levels of organization. For a general
discussion of this organization, see, e.g., Alberts et al., Molecular
Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel,
Biophysical Chemistry Part I: The Conformation of Biological
Macromolecules (1980). "Primary structure" refers to the amino acid
sequence of a particular peptide. "Secondary structure" refers to locally
ordered, three dimensional structures within a polypeptide. These
structures are commonly known as domains. Domains are portions of a
polypeptide that form a compact unit of the polypeptide and are typically
about 18 to 350 amino acids long, e.g., the transmembrane regions, pore
loop domain, and the C-terminal tail domain. Typical domains are made up
of sections of lesser organization such as stretches of β-sheet and
α-helices. "Tertiary structure" refers to the complete three
dimensional structure of a polypeptide monomer. "Quaternary structure"
refers to the three dimensional structure formed by the noncovalent
association of independent tertiary units. Anisotropic terms are also
known as energy terms.

[0102]A "label" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include 32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins for which antisera or monoclonal
antibodies are available (e.g., the polypeptide of SEQ ID NO:2 or 4 can
be made detectable, e.g., by incorporating a radiolabel into the peptide,
and used to detect antibodies specifically reactive with the peptide).

[0103]As used herein a "nucleic acid probe or oligonucleotide" is defined
as a nucleic acid capable of binding to a target nucleic acid of
complementary sequence through one or more types of chemical bonds,
usually through complementary base pairing, usually through hydrogen bond
formation. As used herein, a probe may include natural (i.e., A, G, C, or
T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the
bases in a probe may be joined by a linkage other than a phosphodiester
bond, so long as it does not interfere with hybridization. Thus, for
example, probes may be peptide nucleic acids in which the constituent
bases are joined by peptide bonds rather than phosphodiester linkages. It
will be understood by one of skill in the art that probes may bind target
sequences lacking complete complementarity with the probe sequence
depending upon the stringency of the hybridization conditions. The probes
are preferably directly labeled as with isotopes, chromophores,
lumiphores, chromogens, or indirectly labeled such as with biotin to
which a streptavidin complex may later bind. By assaying for the presence
or absence of the probe, one can detect the presence or absence of the
select sequence or subsequence.

[0104]A "labeled nucleic acid probe or oligonucleotide" is one that is
bound, either covalently, through a linker or a chemical bond, or
noncovalently, through ionic, van der Waals, electrostatic, or hydrogen
bonds to a label such that the presence of the probe may be detected by
detecting the presence of the label bound to the probe.

[0105]The term "recombinant" when used with reference, e.g., to a cell, or
nucleic acid, protein, or vector, indicates that the cell, nucleic acid,
protein or vector, has been modified by the introduction of a
heterologous nucleic acid or protein or the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. Thus, for example, recombinant cells express genes that are not
found within the native (non-recombinant) form of the cell or express
native genes that are otherwise abnormally expressed, under expressed or
not expressed at all.

[0106]A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used herein, a
promoter includes necessary nucleic acid sequences near the start site of
transcription, such as, in the case of a polymerase II type promoter, a
TATA element. A promoter also optionally includes distal enhancer or
repressor elements, which can be located as much as several thousand base
pairs from the start site of transcription. A "constitutive" promoter is
a promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active under
environmental or developmental regulation. The term "operably linked"
refers to a functional linkage between a nucleic acid expression control
sequence (such as a promoter, or array of transcription factor binding
sites) and a second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to the
second sequence.

[0107]The term "heterologous" when used with reference to portions of a
nucleic acid indicates that the nucleic acid comprises two or more
subsequences that are not found in the same relationship to each other in
nature. For instance, the nucleic acid is typically recombinantly
produced, having two or more sequences from unrelated genes arranged to
make a new functional nucleic acid, e.g., a promoter from one source and
a coding region from another source. Similarly, a heterologous protein
indicates that the protein comprises two or more subsequences that are
not found in the same relationship to each other in nature (e.g., a
fusion protein).

[0108]An "expression vector" is a nucleic acid construct, generated
recombinantly or synthetically, with a series of specified nucleic acid
elements that permit transcription of a particular nucleic acid in a host
cell. The expression vector can be part of a plasmid, virus, or nucleic
acid fragment. Typically, the expression vector includes a nucleic acid
to be transcribed operably linked to a promoter.

[0109]The terms "identical" or percent "identity," in the context of two
or more nucleic acids or polypeptide sequences, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of amino acid residues or nucleotides that are the same (i.e.,
60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity
over a specified region of SEQ ID NO:2 or 4), when compared and aligned
for maximum correspondence over a comparison window, or designated region
as measured using one of the following sequence comparison algorithms or
by manual alignment and visual inspection. Such sequences are then said
to be "substantially identical." This definition also refers to the
compliment of a test sequence. Preferably, the identity exists over a
region that is at least about 25 amino acids or nucleotides in length, or
more preferably over a region that is 50-100 amino acids or nucleotides
in length.

[0110]For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary, and
sequence algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference sequence,
based on the program parameters. For sequence comparison of nucleic acids
and proteins to Slo2 or Slo4 nucleic acids and proteins, the BLAST and
BLAST 2.0 algorithms and the default parameters discussed below are used.

[0111]A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected from
the group consisting of from 20 to 600, usually about 50 to about 200,
more usually about 100 to about 150 in which a sequence may be compared
to a reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well-known in the art. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local homology
algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science Dr.,
Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,
Current Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).

[0112]A preferred example of algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410
(1990), respectively. BLAST and BLAST 2.0 are used, with the parameters
described herein, to determine percent sequence identity for the nucleic
acids and proteins of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which either
match or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to as the
neighborhood word score threshold (Altschul et al., supra). These initial
neighborhood word hits act as seeds for initiating searches to find
longer HSPs containing them. The word hits are extended in both
directions along each sequence for as far as the cumulative alignment
score can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for mismatching
residues; always <0). For amino acid sequences, a scoring matrix is
used to calculate the cumulative score. Extension of the word hits in
each direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence is
reached. The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison
of both strands.

[0113]The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul, Proc.
Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match between
two nucleotide or amino acid sequences would occur by chance. For
example, a nucleic acid is considered similar to a reference sequence if
the smallest sum probability in a comparison of the test nucleic acid to
the reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.

[0114]An indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first
nucleic acid is immunologically cross reactive with the antibodies raised
against the polypeptide encoded by the second nucleic acid, as described
below. Thus, a polypeptide is typically substantially identical to a
second polypeptide, for example, where the two peptides differ only by
conservative substitutions. Another indication that two nucleic acid
sequences are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid sequences
are substantially identical is that the same primers can be used to
amplify the sequence.

[0115]The phrase "selectively (or specifically) hybridizes to" refers to
the binding, duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under stringent hybridization conditions when that
sequence is present in a complex mixture (e.g., total cellular or library
DNA or RNA).

[0116]The phrase "stringent hybridization conditions" refers to conditions
under which a probe will hybridize to its target subsequence, typically
in a complex mixture of nucleic acids, but to no other sequences.
Stringent conditions are sequence-dependent and will be different in
different circumstances. Longer sequences hybridize specifically at
higher temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles of
hybridization and the strategy of nucleic acid assays" (1993). Generally,
stringent conditions are selected to be about 5-10° C. lower than
the thermal melting point (Tm) for the specific sequence at a
defined ionic strength pH. The Tm is the temperature (under defined
ionic strength, pH, and nucleic concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent conditions may
also be achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive signal is
at least two times background, preferably 10 times background
hybridization. Exemplary stringent hybridization conditions can be as
following: 50% formamide, 5×SSC, and 1% SDS, incubating at
42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with
wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0117]Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides which
they encode are substantially identical. This occurs, for example, when a
copy of a nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code. In such cases, the nucleic acids typically
hybridize under moderately stringent hybridization conditions. Exemplary
"moderately stringent hybridization conditions" include a hybridization
in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a
wash in 1×SSC at 45° C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize that
alternative hybridization and wash conditions can be utilized to provide
conditions of similar stringency. Additional guidelines for determining
hybridization parameters are provided in numerous reference, e.g., and
Current Protocols in Molecular Biology, ed. Ausubel, et al.

[0118]For PCR, a temperature of about 36° C. is typical for low
stringency amplification, although annealing temperatures may vary
between about 32° C. and 48° C. depending on primer length.
For high stringency PCR amplification, a temperature of about 62°
C. is typical, although high stringency annealing temperatures can range
from about 50° C. to about 65° C., depending on the primer
length and specificity. Typical cycle conditions for both high and low
stringency amplifications include a denaturation phase of 90°
C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2
min., and an extension phase of about 72° C. for 1-2 min.
Protocols and guidelines for low and high stringency amplification
reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A
Guide to Methods and Applications, Academic Press, Inc. N.Y.).

[0119]"Antibody" refers to a polypeptide comprising a framework region
from an immunoglobulin gene or fragments thereof that specifically binds
and recognizes an antigen. The recognized immunoglobulin genes include
the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region
genes, as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn define
the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0120]An exemplary immunoglobulin (antibody) structural unit comprises a
tetramer. Each tetramer is composed of two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kD) and one "heavy" chain
(about 50-70 kD). The N-terminus of each chain defines a variable region
of about 100 to 110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains respectively.

[0121]Antibodies exist, e.g., as intact immunoglobulins or as a number of
well-characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below the
disulfide linkages in the hinge region to produce F(ab)'2, a dimer
of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab)'2 may be reduced under mild conditions to
break the disulfide linkage in the hinge region, thereby converting the
F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially
Fab with part of the hinge region (see Fundamental Immunology (Paul ed.,
3d ed. 1993). While various antibody fragments are defined in terms of
the digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by using
recombinant DNA methodology. Thus, the term antibody, as used herein,
also includes antibody fragments either produced by the modification of
whole antibodies, or those synthesized de novo using recombinant DNA
methodologies (e.g., single chain Fv) or those identified using phage
display libraries (see, e.g., McCafferty et al., Nature 348:552-554
(1990))

[0122]For preparation of monoclonal or polyclonal antibodies, any
technique known in the art can be used (see, e.g., Kohler & Milstein,
Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc. (1985)). Techniques for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies
to polypeptides of this invention. Also, transgenic mice, or other
organisms such as other mammals, may be used to express humanized
antibodies. Alternatively, phage display technology can be used to
identify antibodies and heteromeric Fab fragments that specifically bind
to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554
(1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0123]An "anti-Slo2" or "anti-Slo4" antibody is an antibody or antibody
fragment that specifically binds a polypeptide encoded by a Slo2 or Slo4
gene, cDNA, or a subsequence thereof.

[0124]A "chimeric antibody" is an antibody molecule in which (a) the
constant region, or a portion thereof, is altered, replaced or exchanged
so that the antigen binding site (variable region) is linked to a
constant region of a different or altered class, effector function and/or
species, or an entirely different molecule which confers new properties
to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor,
drug, etc.; or (b) the variable region, or a portion thereof, is altered,
replaced or exchanged with a variable region having a different or
altered antigen specificity.

[0125]The term "immunoassay" is an assay that uses an antibody to
specifically bind an antigen. The immunoassay is characterized by the use
of specific binding properties of a particular antibody to isolate,
target, and/or quantify the antigen.

[0126]The phrase "specifically (or selectively) binds" to an antibody or
"specifically (or selectively) immunoreactive with," when referring to a
protein or peptide, refers to a binding reaction that is determinative of
the presence of the protein in a heterogeneous population of proteins and
other biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two times the
background and do not substantially bind in a significant amount to other
proteins present in the sample. Specific binding to an antibody under
such conditions may require an antibody that is selected for its
specificity for a particular protein. For example, antibodies, e.g.,
polyclonal or monoclonal antibodies, raised to Slo2 or a Slo4, as shown
in SEQ ID NOS:2 or 4, or splice variants, or portions thereof, can be
selected to obtain only those polyclonal antibodies that are specifically
immunoreactive with Slo2 or Slo4 family members and not with other Slo
proteins. This selection may be achieved by subtracting out antibodies
that cross-react with molecules such as other Slo family members. In
addition, antibodies, e.g., polyclonal or monoclonal antibodies, raised
to human Slo2 or human Slo4 polymorphic variants, alleles, and
conservatively modified variants can be selected to obtain only those
antibodies that recognize human Slo2 or human Slo4, but not other Slo2 or
Slo4 orthologs, e.g., rat Slo2 (rat SLACK). A variety of immunoassay
formats may be used to select antibodies specifically immunoreactive with
a particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive with a
protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988)
for a description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity). Typically a specific or
selective reaction will be at least twice background signal or noise and
more typically more than 10 to 100 times background.

[0127]The phrase "selectively associates with" refers to the ability of a
nucleic acid to "selectively hybridize" with another as defined above, or
the ability of an antibody to "selectively (or specifically) bind to a
protein, as defined above.

[0128]By "host cell" is meant a cell that contains an expression vector
and supports the replication or expression of the expression vector. Host
cells may be prokaryotic cells such as E. coli, or eukaryotic cells such
as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the
like, e.g., cultured cells, explants, and cells in vivo.

[0129]"Biological sample" as used herein is a sample of biological tissue
or fluid that contains Slo2 or Slo4 polypeptides or nucleic acid encoding
a Slo2 or Slo4 protein. Such samples include, but are not limited to,
tissue isolated from humans. Biological samples may also include sections
of tissues such as frozen sections taken for histologic purposes. A
biological sample is typically obtained from a eukaryotic organism,
preferably eukaryotes such as fungi, plants, insects, protozoa, birds,
fish, reptiles, and preferably a mammal such as rat, mice, cow, dog,
guinea pig, or rabbit, and most preferably a primate such as chimpanzees
or humans.

III. ISOLATING A GENE ENCODING A SLO2 OR SLO4 POLYPEPTIDE

[0130]A. General Recombinant DNA Methods

[0131]This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of use
in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression:
A Laboratory Manual (1990); and Current Protocols in Molecular Biology
(Ausubel et al., eds., 1994)).

[0132]For nucleic acids, sizes are given in either kilobases (Kb) or base
pairs (bp). These are estimates derived from agarose or acrylamide gel
electrophoresis, from sequenced nucleic acids, or from published DNA
sequences. For proteins, sizes are given in kilodaltons (kD) or amino
acid residue numbers. Proteins sizes are estimated from gel
electrophoresis, from sequenced proteins, from derived amino acid
sequences, or from published protein sequences.

[0133]Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage & Caruthers, Tetrahedron
Letts. 22:1859-1862 (1981), using an automated synthesizer, as described
in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984).
Purification of oligonucleotides is by either native acrylamide gel
electrophoresis or by anion-exchange HPLC as described in Pearson &
Reanier, J. Chrom. 255:137-149 (1983).

[0136]In general, the nucleic acid sequences encoding Slo2 or a Slo4 and
related nucleic acid sequence homologs are cloned from cDNA and genomic
DNA libraries or isolated using amplification techniques with
oligonucleotide primers. For example, Slo2 or a Slo4 sequences are
typically isolated from human nucleic acid (genomic or cDNA) libraries by
hybridizing with a nucleic acid probe or polynucleotide, the sequence of
which can be derived from SEQ ID NOS:1 or 3. A suitable tissue from which
Slo2 or Slo4 RNA and cDNA can be isolated is nervous system tissue such
as whole brain, or any other tissues in which Slo2 or Slo4 is expressed
(see, e.g., FIGS. 4 and 7).

[0137]Amplification techniques using primers can also be used to amplify
and isolate Slo2 or Slo4. The following primers can also be used to
amplify a sequence of human Slo2:

[0139]These primers can be used, e.g., to amplify either the full length
sequence or a probe of one to several hundred nucleotides, which is then
used to screen a library for full-length Slo2 or Slo4.

[0140]Nucleic acids encoding Slo2 or Slo4 family members can also be
isolated from expression libraries using antibodies as probes. Such
polyclonal or monoclonal antibodies can be raised using the sequence of
SEQ ID NO:2 or 4, or an immunogenic portion thereof, e.g., the C-terminal
tail region, located at amino acids 336-1235 for Slo2 and amino acids
323-1135 for Slo4.

[0141]Slo2 and Slo4 polymorphic variants, orthologs, and alleles that are
substantially identical to a conserved region of Slo2 or Slo4 can be
isolated using Slo2 or Slo4 nucleic acid probes and oligonucleotides
under stringent hybridization conditions, by screening libraries.
Alternatively, expression libraries can be used to clone Slo2 or Slo4
polymorphic variants, orthologs, and alleles by detecting expressed
homologs immunologically with antisera or purified antibodies made
against human Slo2 or Slo4 or immunogenic portions thereof (e.g., the
tail (C-terminal) regions of human Slo2 or Slo4), which also recognize
and selectively bind to the Slo2 or Slo4 homolog.

[0142]To make a cDNA library, one should choose a source that is rich in
Slo2 or Slo4 mRNA, e.g., whole brain. The mRNA is then made into cDNA
using reverse transcriptase, ligated into a recombinant vector, and
transfected into a recombinant host for propagation, screening and
cloning. Methods for making and screening cDNA libraries are well known
(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al.,
supra; Ausubel et al., supra).

[0143]For a genomic library, the DNA is extracted from the tissue and
either mechanically sheared or enzymatically digested to yield fragments
of about 12-20 kb. The fragments are then separated by gradient
centrifugation from undesired sizes and are constructed in bacteriophage
lambda vectors. These vectors and phage are packaged in vitro.
Recombinant phage are analyzed by plaque hybridization as described in
Benton & Davis, Science 196:180-182 (1977). Colony hybridization is
carried out as generally described in Grunstein et al., Proc. Natl. Acad.
Sci. USA., 72:3961-3965 (1975).

[0144]An alternative method of isolating Slo2 or Slo4 nucleic acid and its
orthologs, alleles, mutants, polymorphic variants, and conservatively
modified variants combines the use of synthetic oligonucleotide primers
and amplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195
and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis
et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and
ligase chain reaction (LCR) can be used to amplify nucleic acid sequences
of human Slo2 or Slo4 directly from mRNA, from cDNA, from genomic
libraries or cDNA libraries. Degenerate oligonucleotides can be designed
to amplify Slo2 or Slo4 homologs using the sequences provided herein.
Restriction endonuclease sites can be incorporated into the primers.
Polymerase chain reaction or other in vitro amplification methods may
also be useful, for example, to clone nucleic acid sequences that code
for proteins to be expressed, to make nucleic acids to use as probes for
detecting the presence of Slo2 or Slo4 encoding mRNA in physiological
samples, for nucleic acid sequencing, or for other purposes. Genes
amplified by the PCR reaction can be purified from agarose gels and
cloned into an appropriate vector.

[0145]Gene expression of Slo2 or Slo4 can also be analyzed by techniques
known in the art, e.g., reverse transcription and amplification of mRNA,
isolation of total RNA or poly A.sup.+ RNA, northern blotting, dot
blotting, in situ hybridization, RNase protection, high density
polynucleotide array technology and the like.

[0146]Synthetic oligonucleotides can be used to construct recombinant Slo2
or Slo4 genes for use as probes or for expression of protein. This method
is performed using a series of overlapping oligonucleotides usually
40-120 by in length, representing both the sense and nonsense strands of
the gene. These DNA fragments are then annealed, ligated and cloned.
Alternatively, amplification techniques can be used with precise primers
to amplify a specific subsequence of the Slo2 or Slo4 gene. The specific
subsequence is then ligated into an expression vector.

[0149]To obtain high level expression of a cloned gene, such as those
cDNAs encoding Slo2 or Slo4, one typically subclones Slo2 or Slo4 into an
expression vector that contains a strong promoter to direct
transcription, a transcription/translation terminator, and if for a
nucleic acid encoding a protein, a ribosome binding site for
translational initiation. Suitable bacterial promoters are well known in
the art and described, e.g., in Sambrook et al., and Ausubel et al,
supra. Bacterial expression systems for expressing Slo2 or Slo4 protein
are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et
al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983).
Kits for such expression systems are commercially available. Eukaryotic
expression systems for mammalian cells, yeast, and insect cells are well
known in the art and are also commercially available.

[0150]Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application. The
promoter is preferably positioned about the same distance from the
heterologous transcription start site as it is from the transcription
start site in its natural setting. As is known in the art, however, some
variation in this distance can be accommodated without loss of promoter
function.

[0151]In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains all
the additional elements required for the expression of the Slo2 or Slo4
encoding nucleic acid in host cells. A typical expression cassette thus
contains a promoter operably linked to the nucleic acid sequence encoding
Slo2 or Slo4 and signals required for efficient polyadenylation of the
transcript, ribosome binding sites, and translation termination.
Additional elements of the cassette may include enhancers and, if genomic
DNA is used as the structural gene, introns with functional splice donor
and acceptor sites.

[0152]In addition to a promoter sequence, the expression cassette should
also contain a transcription termination region downstream of the
structural gene to provide for efficient termination. The termination
region may be obtained from the same gene as the promoter sequence or may
be obtained from different genes.

[0153]The particular expression vector used to transport the genetic
information into the cell is not particularly critical. Any of the
conventional vectors used for expression in eukaryotic or prokaryotic
cells may be used. Standard bacterial expression vectors include plasmids
such as pBR322 based plasmids, pSKF, pET23D, and fusion expression
systems such as MBP, GST, and LacZ. Epitope tags can also be added to
recombinant proteins to provide convenient methods of isolation, e.g.,
c-myc.

[0155]Expression of proteins from eukaryotic vectors can be also be
regulated using inducible promoters. With inducible promoters, expression
levels are tied to the concentration of inducing agents, such as
tetracycline or ecdysone, by the incorporation of response elements for
these agents into the promoter. Generally, high level expression is
obtained from inducible promoters only in the presence of the inducing
agent; basal expression levels are minimal. Inducible expression vectors
are often chosen if expression of the protein of interest is detrimental
to eukaryotic cells.

[0156]Some expression systems have markers that provide gene amplification
such as thymidine kinase and dihydrofolate reductase. Alternatively, high
yield expression systems not involving gene amplification are also
suitable, such as using a baculovirus vector in insect cells, with a Slo2
or Slo4 encoding sequence under the direction of the polyhedrin promoter
or other strong baculovirus promoters.

[0157]The elements that are typically included in expression vectors also
include a replicon that functions in E. coli, a gene encoding antibiotic
resistance to permit selection of bacteria that harbor recombinant
plasmids, and unique restriction sites in nonessential regions of the
plasmid to allow insertion of eukaryotic sequences. The particular
antibiotic resistance gene chosen is not critical, any of the many
resistance genes known in the art are suitable. The prokaryotic sequences
are preferably chosen such that they do not interfere with the
replication of the DNA in eukaryotic cells, if necessary.

[0159]Any of the well-known procedures for introducing foreign nucleotide
sequences into host cells may be used. These include the use of calcium
phosphate transfection, polybrene, protoplast fusion, electroporation,
biolistics, liposomes, microinjection, plasma vectors, viral vectors and
any of the other well known methods for introducing cloned genomic DNA,
cDNA, synthetic DNA or other foreign genetic material into a host cell
(see, e.g., Sambrook et al., supra). It is only necessary that the
particular genetic engineering procedure used be capable of successfully
introducing at least one gene into the host cell capable of expressing
Slo2 or Slo4.

[0160]After the expression vector is introduced into the cells, the
transfected cells are cultured under conditions favoring expression of
Slo2 or Slo4, which is recovered from the culture using standard
techniques identified below.

IV. PURIFICATION OF SLO2 OR SLO4 POLYPEPTIDES

[0161]Either naturally occurring or recombinant Slo2 or Slo4 can be
purified for use in functional assays. Naturally occurring Slo2 or Slo4
monomers can be purified, e.g., from human tissue such as whole brain or
cerebral cortex and any other source of a Slo2 or Slo4 homolog.
Recombinant Slo2 or Slo4 monomers can be purified from any suitable
expression system.

[0163]A number of procedures can be employed when recombinant Slo2 or Slo4
monomers are being purified. For example, proteins having established
molecular adhesion properties can be reversible fused to the Slo2 or Slo4
monomers. With the appropriate ligand, the Slo2 or Slo4 monomers can be
selectively adsorbed to a purification column and then freed from the
column in a relatively pure form. The fused protein is then removed by
enzymatic activity. Finally the Slo2 or Slo4 monomers could be purified
using immunoaffinity columns.

[0164]A. Purification of Slo2 or Slo4 Monomers from Recombinant Bacteria

[0165]Recombinant proteins are expressed by transformed bacteria in large
amounts, typically after promoter induction; but expression can be
constitutive. Promoter induction with IPTG is one example of an inducible
promoter system. Bacteria are grown according to standard procedures in
the art. Fresh or frozen bacteria cells are used for isolation of
protein.

[0166]Proteins expressed in bacteria may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for purification of
the Slo2 or Slo4 monomers inclusion bodies. For example, purification of
inclusion bodies typically involves the extraction, separation and/or
purification of inclusion bodies by disruption of bacterial cells, e.g.,
by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM
MgCl2, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can
be lysed using 2-3 passages through a French Press, homogenized using a
Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of
lysing bacteria are apparent to those of skill in the art (see, e.g.,
Sambrook et al., supra; Ausubel et al., supra).

[0167]If necessary, the inclusion bodies are solubilized, and the lysed
cell suspension is typically centrifuged to remove unwanted insoluble
matter. Proteins that formed the inclusion bodies may be renatured by
dilution or dialysis with a compatible buffer. Suitable solvents include,
but are not limited to urea (from about 4 M to about 8 M), formamide (at
least about 80%, volume/volume basis), and guanidine hydrochloride (from
about 4 M to about 8 M). Some solvents which are capable of solubilizing
aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70%
formic acid, are inappropriate for use in this procedure due to the
possibility of irreversible denaturation of the proteins, accompanied by
a lack of immunogenicity and/or activity. Although guanidine
hydrochloride and similar agents are denaturants, this denaturation is
not irreversible and renaturation may occur upon removal (by dialysis,
for example) or dilution of the denaturant, allowing re-formation of
immunologically and/or biologically active protein. Other suitable
buffers are known to those skilled in the art. Human Slo monomers are
separated from other bacterial proteins by standard separation
techniques, e.g., with Ni-NTA agarose resin.

[0168]Alternatively, it is possible to purify the Slo2 or Slo4 monomers
from bacteria periplasm. After lysis of the bacteria, when the Slo2 or
Slo4 monomers are exported into the periplasm of the bacteria, the
periplasmic fraction of the bacteria can be isolated by cold osmotic
shock in addition to other methods known to skill in the art. To isolate
recombinant proteins from the periplasm, the bacterial cells are
centrifuged to form a pellet. The pellet is resuspended in a buffer
containing 20% sucrose. To lyse the cells, the bacteria are centrifuged
and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an
ice bath for approximately 10 minutes. The cell suspension is centrifuged
and the supernatant decanted and saved. The recombinant proteins present
in the supernatant can be separated from the host proteins by standard
separation techniques well known to those of skill in the art.

[0171]Often as an initial step, particularly if the protein mixture is
complex, an initial salt fractionation can separate many of the unwanted
host cell proteins (or proteins derived from the cell culture media) from
the recombinant protein of interest. The preferred salt is ammonium
sulfate. Ammonium sulfate precipitates proteins by effectively reducing
the amount of water in the protein mixture. Proteins then precipitate on
the basis of their solubility. The more hydrophobic a protein is, the
more likely it is to precipitate at lower ammonium sulfate
concentrations. A typical protocol includes adding saturated ammonium
sulfate to a protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will precipitate the
most hydrophobic of proteins. The precipitate is then discarded (unless
the protein of interest is hydrophobic) and ammonium sulfate is added to
the supernatant to a concentration known to precipitate the protein of
interest. The precipitate is then solubilized in buffer and the excess
salt removed if necessary, either through dialysis or diafiltration.
Other methods that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can be
used to fractionate complex protein mixtures.

[0172]Size Differential Filtration

[0173]The molecular weight of the Slo2 or Slo4 monomers can be used to
isolate it from proteins of greater and lesser size using ultrafiltration
through membranes of different pore size (for example, Amicon or
Millipore membranes). As a first step, the protein mixture is
ultrafiltered through a membrane with a pore size that has a lower
molecular weight cut-off than the molecular weight of the protein of
interest. The retentate of the ultrafiltration is then ultrafiltered
against a membrane with a molecular cut off greater than the molecular
weight of the protein of interest. The recombinant protein will pass
through the membrane into the filtrate. The filtrate can then be
chromatographed as described below.

[0174]Column Chromatography

[0175]The Slo2 or Slo4 monomers can also be separated from other proteins
on the basis of its size, net surface charge, hydrophobicity, and
affinity for ligands. In addition, antibodies raised against proteins can
be conjugated to column matrices and the proteins immunopurified. All of
these methods are well known in the art. It will be apparent to one of
skill that chromatographic techniques can be performed at any scale and
using equipment from many different manufacturers (e.g., Pharmacia
Biotech).

V. IMMUNOLOGICAL DETECTION OF SLO2 OR SLO4 POLYPEPTIDES

[0176]In addition to the detection of Slo2 or Slo4 genes and gene
expression using nucleic acid hybridization technology, one can also use
immunoassays to detect the Slo2 or Slo4 monomers of the invention.
Immunoassays can be used to qualitatively or quantitatively analyze the
hSlo2 or Slo4 monomers. A general overview of the applicable technology
can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

[0177]A. Antibodies to Slo2 or Slo4 Monomers

[0178]Methods of producing polyclonal and monoclonal antibodies that react
specifically with the Slo2 or Slo4 monomers, or Slo2 or Slo4 monomers
from particular species such as human Slo2 or Slo4 are known to those of
skill in the art (see, e.g., Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles
and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497
(1975). Such techniques include antibody preparation by selection of
antibodies from libraries of recombinant antibodies in phage or similar
vectors, as well as preparation of polyclonal and monoclonal antibodies
by immunizing rabbits or mice (see, e.g., Huse et al., Science
246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).

[0179]A number of immunogens comprising portions of Slo2 or Slo4 monomers
may be used to produce antibodies specifically reactive with Slo2 or Slo4
monomers. For example, recombinant Slo2 or Slo4 monomers or an antigenic
fragment thereof, such as a conserved region (see, e.g., the pore loop or
the C-terminal tail domains), can be isolated as described herein.
Recombinant protein can be expressed in eukaryotic or prokaryotic cells
as described above, and purified as generally described above.
Recombinant protein is the preferred immunogen for the production of
monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide
derived from the sequences disclosed herein and conjugated to a carrier
protein can be used an immunogen. Naturally occurring protein may also be
used either in pure or impure form. The product is then injected into an
animal capable of producing antibodies. Either monoclonal or polyclonal
antibodies may be generated, for subsequent use in immunoassays to
measure the protein.

[0180]Methods of production of polyclonal antibodies are known to those of
skill in the art. An inbred strain of mice (e.g., BALB/C mice) or rabbits
is immunized with the protein using a standard adjuvant, such as Freund's
adjuvant, and a standard immunization protocol. The animal's immune
response to the immunogen preparation is monitored by taking test bleeds
and determining the titer of reactivity to the beta subunits. When
appropriately high titers of antibody to the immunogen are obtained,
blood is collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to the
protein can be done if desired (see, Harlow & Lane, supra).

[0181]Monoclonal antibodies may be obtained by various techniques familiar
to those skilled in the art. Briefly, spleen cells from an animal
immunized with a desired antigen are immortalized, commonly by fusion
with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519
(1976)). Alternative methods of immortalization include transformation
with Epstein Barr Virus, oncogenes, or retroviruses, or other methods
well known in the art. Colonies arising from single immortalized cells
are screened for production of antibodies of the desired specificity and
affinity for the antigen, and yield of the monoclonal antibodies produced
by such cells may be enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one may
isolate DNA sequences which encode a monoclonal antibody or a binding
fragment thereof by screening a DNA library from human B cells according
to the general protocol outlined by Huse, et al., Science 246:1275-1281
(1989).

[0182]Monoclonal antibodies and polyclonal sera are collected and titered
against the immunogen protein in an immunoassay, for example, a solid
phase immunoassay with the immunogen immobilized on a solid support.
Typically, polyclonal antisera with a titer of 104 or greater are
selected and tested for their cross reactivity against non-Slo family
proteins and other Slo family proteins, using a competitive binding
immunoassay. Specific polyclonal antisera and monoclonal antibodies will
usually bind with a Kd of at least about 0.1 mM, more usually at
least about 1 μM, preferably at least about 0.1 μM or better, and
most preferably, 0.01 μM or better. Antibodies specific only for a
particular Slo2 or Slo4 ortholog, such as human Slo2 or Slo4, can also be
made, by subtracting out other cross-reacting orthologs from a species
such as a non-human mammal.

[0183]Once the specific antibodies against Slo2 or Slo4 are available, the
polypeptides can be detected by a variety of immunoassay methods. For a
review of immunological and immunoassay procedures, see Basic and
Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover,
the immunoassays of the present invention can be performed in any of
several configurations, which are reviewed extensively in Enzyme
Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

[0184]B. Immunological Binding Assays

[0185]The Slo2 or Slo4 polypeptides of the invention can be detected
and/or quantified using any of a number of well recognized immunological
binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168). For a review of the general immunoassays, see
also Methods in Cell Biology: Antibodies in Cell Biology, volume 37
(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds.,
7th ed. 1991). Immunological binding assays (or immunoassays)
typically use an antibody that specifically binds to a protein or antigen
of choice (in this case Slo2 or Slo4 or an antigenic subsequence
thereof). The antibody (e.g., anti-Slo2 or Slo4) may be produced by any
of a number of means well known to those of skill in the art and as
described above.

[0186]Immunoassays also often use a labeling agent to specifically bind to
and label the complex formed by the antibody and antigen. The labeling
agent may itself be one of the moieties comprising the antibody/antigen
complex. Thus, the labeling agent may be a labeled Slo2 or Slo4
polypeptide or a labeled anti-Slo2 or Slo4 antibody. Alternatively, the
labeling agent may be a third moiety, such a secondary antibody, which
specifically binds to the antibody/Slo2 or Slo4 complex (a secondary
antibody is typically specific to antibodies of the species from which
the first antibody is derived). Other proteins capable of specifically
binding immunoglobulin constant regions, such as protein A or protein G
may also be used as the label agent. These proteins exhibit a strong
non-immunogenic reactivity with immunoglobulin constant regions from a
variety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406
(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). The labeling
agent can be modified with a detectable moiety, such as biotin, to which
another molecule can specifically bind, such as streptavidin. A variety
of detectable moieties are well known to those skilled in the art.

[0187]Throughout the assays, incubation and/or washing steps may be
required after each combination of reagents. Incubation steps can vary
from about 5 seconds to several hours, preferably from about 5 minutes to
about 24 hours. However, the incubation time will depend upon the assay
format, antigen, volume of solution, concentrations, and the like.
Usually, the assays will be carried out at ambient temperature, although
they can be conducted over a range of temperatures, such as 10° C.
to 40° C.

[0188]Non-Competitive Assay Formats

[0189]Immunoassays for detecting the Slo2 or Slo4 in samples may be either
competitive or noncompetitive. Noncompetitive immunoassays are assays in
which the amount of antigen is directly measured. In one preferred
"sandwich" assay, for example, the anti-Slo2 or Slo4 subunit antibodies
can be bound directly to a solid substrate on which they are immobilized.
These immobilized antibodies then capture Slo2 or Slo4 present in the
test sample. The Slo2 or Slo4 monomers are thus immobilized and then
bound by a labeling agent, such as a second Slo2 or Slo4 antibody bearing
a label. Alternatively, the second antibody may lack a label, but it may,
in turn, be bound by a labeled third antibody specific to antibodies of
the species from which the second antibody is derived. The second or
third antibody is typically modified with a detectable moiety, such as
biotin, to which another molecule specifically binds, e.g., streptavidin,
to provide a detectable moiety.

[0190]Competitive Assay Formats

[0191]In competitive assays, the amount of the Slo2 or Slo4 present in the
sample is measured indirectly by measuring the amount of known, added
(exogenous) Slo2 or Slo4 displaced (competed away) from an anti-Slo2 or
Slo4 antibody by the unknown Slo2 or Slo4 present in a sample. In one
competitive assay, a known amount of the Slo2 or Slo4 is added to a
sample and the sample is then contacted with an antibody that
specifically binds to the Slo2 or Slo4. The amount of exogenous Slo2 or
Slo4 bound to the antibody is inversely proportional to the concentration
of the Slo2 or Slo4 present in the sample. In a particularly preferred
embodiment, the antibody is immobilized on a solid substrate. The amount
of Slo2 or Slo4 bound to the antibody may be determined either by
measuring the amount of Slo2 or Slo4 present in a Slo2 or Slo4/antibody
complex, or alternatively by measuring the amount of remaining
uncomplexed protein. The amount of Slo2 or Slo4 may be detected by
providing a labeled Slo2 or Slo4 molecule.

[0192]A hapten inhibition assay is another preferred competitive assay. In
this assay the known Slo2 or Slo4 is immobilized on a solid substrate. A
known amount of anti-Slo2 or Slo4 antibody is added to the sample, and
the sample is then contacted with the immobilized Slo2 or Slo4. The
amount of anti-Slo2 or Slo4 antibody bound to the known immobilized Slo2
or Slo4 is inversely proportional to the amount of Slo2 or Slo4 present
in the sample. Again, the amount of immobilized antibody may be detected
by detecting either the immobilized fraction of antibody or the fraction
of the antibody that remains in solution. Detection may be direct where
the antibody is labeled or indirect by the subsequent addition of a
labeled moiety that specifically binds to the antibody as described
above.

[0193]Cross-Reactivity Determinations

[0194]Immunoassays in the competitive binding format can also be used for
crossreactivity determinations for Slo2 or Slo4. For example, a Slo2 or
Slo4 protein at least partially corresponding to an amino acid sequence
of SEQ ID NO:2 or 4 or an immunogenic region thereof, such as a conserved
region (e.g., the pore loop or tail domain), can be immobilized to a
solid support. Other proteins such as other Slo family members are added
to the assay so as to compete for binding of the antisera to the
immobilized antigen. The ability of the added proteins to compete for
binding of the antisera to the immobilized protein is compared to the
ability of the Slo2 or Slo4 or immunogenic portion thereof to compete
with itself. The percent crossreactivity for the above proteins is
calculated, using standard calculations. Those antisera with less than
10% crossreactivity with each of the added proteins listed above are
selected and pooled. The cross-reacting antibodies are optionally removed
from the pooled antisera by immunoabsorption with the added considered
proteins, e.g., distantly related homologs. Antibodies that specifically
bind only to Slo2 or Slo4, or only to particular orthologs of Slo2 or
Slo4, such as human Slo2 or Slo4, can also be made using this
methodology.

[0195]The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a second
protein, thought to be perhaps Slo2 or Slo4 or an allele, ortholog, or
polymorphic variant thereof, to the immunogen protein. In order to make
this comparison, the two proteins are each assayed at a wide range of
concentrations and the amount of each protein required to inhibit 50% of
the binding of the antisera to the immobilized protein is determined. If
the amount of the second protein required to inhibit 50% of binding is
less than 10 times the amount of the protein encoded by Slo2 or Slo4 that
is required to inhibit 50% of binding, then the second protein is said to
specifically bind to the polyclonal antibodies generated to the
respective Slo2 or Slo4 immunogen.

[0196]Other Assay Formats

[0197]Western blot (immunoblot) analysis is used to detect and quantify
the presence of the Slo2 or Slo4 in the sample. The technique generally
comprises separating sample proteins by gel electrophoresis on the basis
of molecular weight, transferring the separated proteins to a suitable
solid support, (such as a nitrocellulose filter, a nylon filter, or
derivatized nylon filter), and incubating the sample with the antibodies
that specifically bind Slo2 or Slo4. The anti-Slo2 or Slo4 antibodies
specifically bind to Slo2 or Slo4 on the solid support. These antibodies
may be directly labeled or alternatively may be subsequently detected
using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that
specifically bind to the anti-Slo2 or Slo4 antibodies.

[0198]Other assay formats include liposome immunoassays (LIA), which use
liposomes designed to bind specific molecules (e.g., antibodies) and
release encapsulated reagents or markers. The released chemicals are then
detected according to standard techniques (see, Monroe et al., Amer.
Clin. Prod. Rev. 5:34-41 (1986)).

[0199]Reduction of Non-Specific Binding

[0200]One of skill in the art will appreciate that it is often desirable
to minimize non-specific binding in immunoassays. Particularly, where the
assay involves an antigen or antibody immobilized on a solid substrate it
is desirable to minimize the amount of non-specific binding to the
substrate. Means of reducing such non-specific binding are well known to
those of skill in the art. Typically, this technique involves coating the
substrate with a proteinaceous composition. In particular, protein
compositions such as bovine serum albumin (BSA), nonfat powdered milk,
and gelatin are widely used with powdered milk being most preferred.

[0201]Labels

[0202]The particular label or detectable group used in the assay is not a
critical aspect of the invention, as long as it does not significantly
interfere with the specific binding of the antibody used in the assay.
The detectable group can be any material having a detectable physical or
chemical property. Such detectable labels have been well-developed in the
field of immunoassays and, in general, most any label useful in such
methods can be applied to the present invention. Thus, a label is any
composition detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. Useful labels in
the present invention include magnetic beads (e.g., DYNABEADS®),
fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine,
and the like), radiolabels (e.g., 3H, 125I, 35S, 14C,
or 32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic beads (e.g.,
polystyrene, polypropylene, latex, etc.).

[0203]The label may be coupled directly or indirectly to the desired
component of the assay according to methods well known in the art. As
indicated above, a wide variety of labels may be used, with the choice of
label depending on sensitivity required, ease of conjugation with the
compound, stability requirements, available instrumentation, and disposal
provisions.

[0204]Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to the
molecule. The ligand then binds to another molecule (e.g., streptavidin),
which is either inherently detectable or covalently bound to a signal
system, such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound. The ligands and their targets can be used in
any suitable combination with antibodies that recognize hSlo2 or Slo4, or
secondary antibodies that recognize anti-hSlo2 or Slo4 antibodies.

[0205]The molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of
interest as labels will primarily be hydrolases, particularly
phosphatases, esterases and glycosidases, or oxidases, particularly
peroxidases. Fluorescent compounds include fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
Chemiluminescent compounds include luciferin, and
2,3-dihydrophthalazinediones, e.g., luminol. For a review of various
labeling or signal producing systems that may be used, see, U.S. Pat. No.
4,391,904.

[0206]Means of detecting labels are well known to those of skill in the
art. Thus, for example, where the label is a radioactive label, means for
detection include a scintillation counter or photographic film as in
autoradiography. Where the label is a fluorescent label, it may be
detected by exciting the fluorochrome with the appropriate wavelength of
light and detecting the resulting fluorescence. The fluorescence may be
detected visually, by means of photographic film, by the use of
electronic detectors such as charge coupled devices (CCDs) or
photomultipliers and the like. Similarly, enzymatic labels may be
detected by providing the appropriate substrates for the enzyme and
detecting the resulting reaction product. Finally, simple colorimetric
labels may be detected simply by observing the color associated with the
label. Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the bead.

[0207]Some assay formats do not require the use of labeled components. For
instance, agglutination assays can be used to detect the presence of the
target antibodies. In this case, antigen-coated particles are
agglutinated by samples comprising the target antibodies. In this format,
none of the components need be labeled and the presence of the target
antibody is detected by simple visual inspection.

[0211]Furthermore, such assays can be used to test for inhibitors and
activators of channels comprising Slo2 or Slo4. Such modulators of a
potassium channel are useful for treating various disorders involving
potassium channels. Such modulators of potassium channel activity are
useful for treating disorders, including CNS disorders, such as
neuropathic pain, epilepsy and other seizure disorders, migraines,
anxiety, psychotic disorders such as schizophrenia, bipolar disease, and
depression. Such modulators are also useful as neuroprotective agents
(e.g., to prevent stroke). Modulators could also be useful in treating
cognitive disorders of learning and memory caused by diseases such as
Alzheimer's, or to enhance learning and memory in the aging population,
as well providing neuroprotection. Finally, such modulators could be
useful for treating hypercontractility of muscles, cardiac arrhythmias,
inflammation, asthma, and as immunosuppressants or stimulants.

[0212]Such modulators are also useful for investigation of the channel
diversity provided by Slo2 or Slo4 and the regulation/modulation of
potassium channel activity provided by Slo2 or Slo4.

[0213]Modulators of the Slo potassium channels are tested using
biologically active Slo2 or Slo4, either recombinant or naturally
occurring, preferably human Slo2 or Slo4. Slo2 or Slo4 can be isolated,
co-expressed or expressed in a cell, or expressed in a membrane derived
from a cell. In such assays, Slo2 or Slo4 is expressed alone to form a
homomeric potassium channel or is co-expressed with a second alpha
subunit (e.g., another Slo family member, e.g., Slo1 or Slo3) so as to
form a heteromeric potassium channel. Slo2 or Slo4 polypeptides can also
be expressed with additional beta subunits. Modulation is tested using
one of the in vitro or in vivo assays described herein.

[0214]Samples or assays that are treated with a potential potassium
channel inhibitor or activator are compared to control samples without
the test compound, to examine the extent of modulation. Control samples
(untreated with activators or inhibitors) are assigned a relative
potassium channel activity value of 100. Inhibition of channels
comprising a Slo2 or Slo4 polypeptide is achieved when the potassium
channel activity value relative to the control is about 90%, preferably
50%, more preferably 25%. Activation of channels comprising a Slo2 or
Slo4 polypeptide is achieved when the potassium channel activity value
relative to the control is 110%, more preferably 150%, more preferable
200% higher. Compounds that increase the flux of ions will cause a
detectable increase in the ion current density by increasing the
probability of a channel comprising a Slo2 or Slo4 polypeptide being
open, by decreasing the probability of it being closed, by increasing
conductance through the channel, and/or by allowing the passage of ions.

[0215]Preferably, the Slo2 or Slo4 polypeptide used in the assay will have
the sequence displayed in SEQ ID NO:2 or 4 or a conservatively modified
variant thereof. Alternatively, the Slo2 or Slo4 of the assay will be
derived from a eukaryote and include an amino acid subsequence having
substantial amino acid sequence identity to a conserved region (see,
e.g., pore loop or tail domain) of human Slo2 or Slo4. Generally, the
amino acid sequence identity will be at least 60%, preferably at least
65%, 70%, 75%, 80%, 85%, or 90%, most preferably at least 95% or higher.

[0216]In Vitro Assays

[0217]Assays to identify compounds with potassium channel modulating
activity can be performed in vitro, e.g., binding assays. Purified
recombinant or naturally occurring Slo2 or Slo4 protein, or a channel
comprising Slo 2 or Slo4 protein, can be used in the in vitro methods of
the invention. In addition to purified Slo2 or Slo4 protein or channel
comprising the same, the recombinant or naturally occurring Slo2 or Slo4
protein can be part of a cellular lysate or a cell membrane. As described
below, the assay can be either solid state or soluble. Preferably, the
protein or membrane is bound to a solid support, either covalently or
non-covalently. Often, the in vitro assays of the invention are ligand or
toxin binding or ligand affinity assays, either non-competitive or
competitive. Other in vitro assays include measuring changes in
spectroscopic (e.g., fluorescence, absorbance, refractive index),
hydrodynamic (e.g., shape), chromatographic, or solubility properties for
the protein or channel. Cell membranes or lysates can also be used to
measure changes in polarization (i.e., electrical potential) of the cell
or membrane expressing the potassium channel comprising a Slo2 or Slo4
polypeptide, as described below.

[0218]In Vivo Assays

[0219]In another embodiment, Slo2 or Slo4 protein is expressed in a cell,
and functional, e.g., physical and chemical or phenotypic, changes are
assayed to identify potassium channel modulators. For example, using
cell- or animal based assays, changes in ion flux may be assessed by
determining changes in polarization (i.e., electrical potential) of the
cell or membrane expressing the potassium channel comprising a Slo2 or
Slo4 polypeptide. A preferred means to determine changes in cellular
polarization is by measuring changes in current (thereby measuring
changes in polarization) with voltage-clamp and patch-clamp techniques,
e.g., the "cell-attached" mode, the "inside-out" mode, and the "whole
cell" mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595
(1997)). Whole cell currents are conveniently determined using the
standard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85
(1981). Other known assays include: radiolabeled rubidium flux assays and
fluorescence assays using voltage-sensitive dyes or ion sensitive dyes
(see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988);
Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al.,
J. Membrane Biology 137:59-70 (1994)). Assays for compounds capable of
inhibiting or increasing potassium flux through the channel proteins
comprising a Slo2 or Slo4 polypeptide can be performed by application of
the compounds to a bath solution in contact with and comprising cells
having a channel of the present invention (see, e.g., Blatz et al.,
Nature 323:718-720 (1986); Park, J. Physiol. 481:555-570 (1994)).
Generally, the compounds to be tested are present in the range from 1
μM to 100 mM.

[0220]The effects of the test compounds upon the function of the channels
can be measured by changes in the electrical currents or ionic flux or by
the consequences of changes in currents and flux. Changes in electrical
current or ionic flux are measured by either increases or decreases in
flux of ions such as potassium or rubidium ions. The ions can be measured
in a variety of standard ways. They can be measured directly by
concentration changes of the ions, e.g., changes in intracellular
concentrations, or indirectly by membrane potential or by radio-labeling
of the ions. Consequences of the test compound on ion flux can be quite
varied. Accordingly, any suitable physiological change can be used to
assess the influence of a test compound on the channels of this
invention.

[0221]For example, the effects of a test compound can be measured by a
ligand or toxin binding assay. One can also measure a variety of effects
such as transmitter release (e.g., dopamine), intracellular calcium
changes, hormone release (e.g., insulin), transcriptional changes to both
known and uncharacterized genetic markers (e.g., northern blots), cell
volume changes (e.g., in red blood cells), immunoresponses (e.g., T cell
activation), changes in cell metabolism such as cell growth or pH
changes, and changes in intracellular second messengers such as cyclic
nucleotides.

[0222]Slo2 or Slo4 orthologs, alleles, polymorphic variants, and
conservatively modified variants will generally confer substantially
similar properties on a channel comprising a Slo2 or Slo4 polypeptide, as
described above. In a preferred embodiment, the cell placed in contact
with a compound that is suspected to be a Slo2 or Slo4 homolog is assayed
for increasing or decreasing ion flux in a eukaryotic cell, e.g., an
oocyte of Xenopus (e.g., Xenopus laevis) or a mammalian cell such as a
CHO or HeLa cell. Channels that are affected by compounds in ways similar
to Slo2 or Slo4 are considered homologs or orthologs of Slo2 or Slo4.

[0223]Animal Models

[0224]Animal models also find use in screening for potassium channel
modulators. Transgenic animal technology, including gene knockout
technology as a result of homologous recombination with an appropriate
gene targeting vector, or gene overexpression, will result in the absence
or increased expression of the Slo2 or Slo4 protein. When desired,
tissue-specific expression or knockout of the Slo2 or Slo4 protein may be
necessary. Transgenic animals generated by such methods find use as
animal models of abnormal ion flux and are additionally useful in
screening for modulators of potassium channels.

[0225]B. Modulators

[0226]The compounds tested as modulators of Slo channels comprising a Slo2
or Slo4 subunit can be any small organic compound, or a biological
entity, such as a protein, e.g., a peptide or antibody, sugar, nucleic
acid, e.g., an antisense molecule, or lipid. Alternatively, modulators
can be genetically altered versions of a Slo2 or Slo4 subunit. Typically,
test compounds will be small organic molecules, antibodies, antisense
molecules, and peptides. Essentially any chemical compound can be used as
a potential modulator or ligand in the assays of the invention, although
most often compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions are used. The assays are designed to screen large
chemical libraries by automating the assay steps and providing compounds
from any convenient source to assays, which are typically run in parallel
(e.g., in microtiter formats on microtiter plates in robotic assays). It
will be appreciated that there are many suppliers of chemical compounds,
including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich
(St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland)
and the like.

[0227]In one preferred embodiment, high throughput screening methods
involve providing a combinatorial chemical or peptide library containing
a large number of potential therapeutic compounds (potential modulator or
ligand compounds). Such "combinatorial chemical libraries" or "ligand
libraries" are then screened in one or more assays, as described herein,
to identify those library members (particular chemical species or
subclasses) that display a desired characteristic activity. The compounds
thus identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.

[0228]A combinatorial chemical library is a collection of diverse chemical
compounds generated by either chemical synthesis or biological synthesis,
by combining a number of chemical "building blocks" such as reagents. For
example, a linear combinatorial chemical library such as a polypeptide
library is formed by combining a set of chemical building blocks (amino
acids) in every possible way for a given compound length (i.e., the
number of amino acids in a polypeptide compound). Millions of chemical
compounds can be synthesized through such combinatorial mixing of
chemical building blocks.

[0231]In one embodiment, the invention provides solid phase based in vitro
assays in a high throughput format, where the cell or tissue expressing a
Slo channel comprising a human Slo2 or Slo4 subunit is attached to a
solid phase substrate. In the high throughput assays of the invention, it
is possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter plate
can be used to run a separate assay against a selected potential
modulator, or, if concentration or incubation time effects are to be
observed, every 5-10 wells can test a single modulator. Thus, a single
standard microtiter plate can assay about 96 modulators. If 1536 well
plates are used, then a single plate can easily assay from about
100-about 1500 different compounds. It is possible to assay many plates
per day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000
or more different compounds are possible using the integrated systems of
the invention.

[0232]C. Solid State and Soluble High Throughput Assays

[0233]In one embodiment the invention provides high throughput soluble
assays using, e.g., isolated potassium channels comprising a Slo2 or Slo4
polypeptide; a membrane comprising a Slo2 or Slo4 potassium channel, or a
cell or tissue expressing potassium channels comprising a Slo2 or Slo4
polypeptide, either naturally occurring or recombinant. In another
embodiment, the invention provides solid phase based in vitro assays in a
high throughput format, where Slo2 or Slo4 potassium channel attached to
a solid phase substrate. In another assay, the cell membrane or cell
comprising the channel can be attached to a solid phase substrate. In yet
another embodiment, the test compound is attached to a solid phase
substrate.

[0234]In the high throughput assays of the invention, it is possible to
screen thousands of different modulators or ligands in a single day. In
particular, each well of a microtiter plate can be used to run a separate
assay against a selected potential modulator, or, if concentration or
incubation time effects are to be observed, every 5-10 wells can test a
single modulator. Thus, a single standard microtiter plate can assay
about 100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100-about 1500 different
compounds. It is possible to assay many plates per day; assay screens for
up to about 6,000, 20,000, 50,000, or more than 100,000 different
compounds are possible using the integrated systems of the invention.

[0235]The channel of interest, or a cell or membrane comprising the
channel of interest can be bound to the solid state component, directly
or indirectly, via covalent or non covalent linkage e.g., via a tag. The
tag can be any of a variety of components. In general, a molecule which
binds the tag (a tag binder) is fixed to a solid support, and the tagged
molecule of interest (e.g., the taste transduction molecule of interest)
is attached to the solid support by interaction of the tag and the tag
binder.

[0236]A number of tags and tag binders can be used, based upon known
molecular interactions well described in the literature. For example,
where a tag has a natural binder, for example, biotin, protein A, or
protein G, it can be used in conjunction with appropriate tag binders
(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,
etc.) Antibodies to molecules with natural binders such as biotin are
also widely available and appropriate tag binders; see, SIGMA
Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

[0237]Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder pair.
Thousands of specific antibodies are commercially available and many
additional antibodies are described in the literature. For example, in
one common configuration, the tag is a first antibody and the tag binder
is a second antibody which recognizes the first antibody. In addition to
antibody-antigen interactions, receptor-ligand interactions are also
appropriate as tag and tag-binder pairs. For example, agonists and
antagonists of cell membrane receptors (e.g., cell receptor-ligand
interactions such as transferrin, c-kit, viral receptor ligands, cytokine
receptors, chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherein family, the integrin family, the
selectin family, and the like; see, e.g., Pigott & Power, The Adhesion
Molecule Facts Book I (1993). Similarly, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), intracellular
receptors (e.g. which mediate the effects of various small ligands,
including steroids, thyroid hormone, retinoids and vitamin D; peptides),
drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer
configurations), oligosaccharides, proteins, phospholipids and antibodies
can all interact with various cell receptors.

[0238]Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene
sulfides, polysiloxanes, polyimides, and polyacetates can also form an
appropriate tag or tag binder. Many other tag/tag binder pairs are also
useful in assay systems described herein, as would be apparent to one of
skill upon review of this disclosure.

[0239]Common linkers such as peptides, polyethers, and the like can also
serve as tags, and include polypeptide sequences, such as poly gly
sequences of between about 5 and 200 amino acids. Such flexible linkers
are known to persons of skill in the art. For example, poly(ethylene
glycol) linkers are available from Shearwater Polymers, Inc. Huntsville,
Ala. These linkers optionally have amide linkages, sulfhydryl linkages,
or heterofunctional linkages.

[0240]Tag binders are fixed to solid substrates using any of a variety of
methods currently available. Solid substrates are commonly derivatized or
functionalized by exposing all or a portion of the substrate to a
chemical reagent which fixes a chemical group to the surface which is
reactive with a portion of the tag binder. For example, groups which are
suitable for attachment to a longer chain portion would include amines,
hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and
hydroxyalkylsilanes can be used to functionalize a variety of surfaces,
such as glass surfaces. The construction of such solid phase biopolymer
arrays is well described in the literature. See, e.g., Merrifield, J. Am.
Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank & Doring,
Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide
sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991);
Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et
al., Nature Medicine 2(7):753759 (1996) (all describing arrays of
biopolymers fixed to solid substrates). Non-chemical approaches for
fixing tag binders to substrates include other common methods, such as
heat, cross-linking by UV radiation, and the like.

VII. COMPUTER ASSISTED DRUG DESIGN USING SLO2 OR SLO4

[0241]Yet another assay for compounds that modulate the activities of a
Slo2 or Slo4 channel involves computer assisted drug design, in which a
computer system is used to generate a three-dimensional structure of Slo2
or Slo4 based on the structural information encoded by the amino acid
sequence. The input amino acid sequence interacts directly and actively
with a pre-established algorithm in a computer program to yield
secondary, tertiary, and quaternary structural models of the protein. The
models of the protein structure are then examined to identify regions of
the structure that have the ability to bind, e.g., ligands or other
potassium channel subunits. These regions are then used to identify
ligands that bind to the protein or region where Slo2 or Slo4 interacts
with other potassium channel subunits.

[0242]The three-dimensional structural model of the protein is generated
by entering channel protein amino acid sequences of at least 25, 50, 75
or 100 or more amino acid residues or corresponding nucleic acid
sequences encoding a Slo2 or Slo4 monomer into the computer system. The
amino acid sequence of each of the monomers is selected from the group
consisting of SEQ ID NO:2 or 4 and a conservatively modified versions
thereof, or an immunogenic portion thereof comprising a conserved region,
e.g., the pore loop or the tail domains. The amino acid sequence
represents the primary sequence or subsequence of each of the proteins,
which encodes the structural information of the protein. At least 25, 50,
75, or 100 residues of the amino acid sequence (or a nucleotide sequence
encoding at least about 25, 50, 75 or 100 amino acids) are entered into
the computer system from computer keyboards, computer readable substrates
that include, but are not limited to, electronic storage media (e.g.,
magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,
CD ROM), information distributed by internet sites, and by RAM. The
three-dimensional structural model of the channel protein is then
generated by the interaction of the amino acid sequence and the computer
system, using software known to those of skill in the art. The resulting
three-dimensional computer model can then be saved on a computer readable
substrate.

[0243]The amino acid sequence represents a primary structure that encodes
the information necessary to form the secondary, tertiary and quaternary
structure of the monomer and the heteromeric potassium channel protein
comprising four monomers. The software looks at certain parameters
encoded by the primary sequence to generate the structural model. These
parameters are referred to as "energy terms," or anisotropic terms and
primarily include electrostatic potentials, hydrophobic potentials,
solvent accessible surfaces, and hydrogen bonding. Secondary energy terms
include van der Waals potentials. Biological molecules form the
structures that minimize the energy terms in a cumulative fashion. The
computer program is therefore using these terms encoded by the primary
structure or amino acid sequence to create the secondary structural
model.

[0244]The tertiary structure of the protein encoded by the secondary
structure is then formed on the basis of the energy terms of the
secondary structure. The user at this point can enter additional
variables such as whether the protein is membrane bound or soluble, its
location in the body, and its cellular location, e.g., cytoplasmic,
surface, or nuclear. These variables along with the energy terms of the
secondary structure are used to form the model of the tertiary structure.
In modeling the tertiary structure, the computer program matches
hydrophobic faces of secondary structure with like, and hydrophilic faces
of secondary structure with like.

[0245]Once the structure has been generated, potential ligand binding
regions are identified by the computer system. Three-dimensional
structures for potential ligands are generated by entering amino acid or
nucleotide sequences or chemical formulas of compounds, as described
above. The three-dimensional structure of the potential ligand is then
compared to that of the Slo2 or Slo4 protein to identify ligands that
bind to Slo2 or Slo4. Binding affinity between the protein and ligands is
determined using energy terms to determine which ligands have an enhanced
probability of binding to the protein.

[0246]Computer systems are also used to screen for mutations, polymorphic
variants, alleles and interspecies homologs of Slo2 or Slo4 genes. Such
mutations can be associated with disease states. Once the variants are
identified, diagnostic assays can be used to identify patients having
such mutated genes associated with disease states. Identification of the
mutated Slo2 or Slo4 genes involves receiving input of a first nucleic
acid, e.g., SEQ ID NOS:1 or 3, or an amino acid sequence encoding Slo2 or
Slo4, e.g., SEQ ID NO:2 or 4, and conservatively modified versions
thereof, or an amino acid sequence comprising a conserved region, e.g.,
the pore loop or the tail domain. The sequence is entered into the
computer system as described above. The first nucleic acid or amino acid
sequence is then compared to a second nucleic acid or amino acid sequence
that has substantial identity to the first sequence. The second sequence
is entered into the computer system in the manner described above. Once
the first and second sequences are compared, nucleotide or amino acid
differences between the sequences are identified. Such sequences can
represent allelic differences in Slo2 or Slo4 genes, preferably human
Slo2 or Slo4 genes and mutations associated with disease states. The
first and second sequences described above can be saved on a computer
readable substrate.

[0248]The present invention provides the nucleic acids of Slo2 or Slo4 for
the transfection of cells in vitro and in vivo. These nucleic acids can
be inserted into any of a number of well-known vectors for the
transfection of target cells and organisms as described below. The
nucleic acids are transfected into cells, ex vivo or in vivo, through the
interaction of the vector and the target cell. The nucleic acid for Slo2
or Slo4, under the control of a promoter, then expresses a Slo2 or Slo4
monomer of the present invention, thereby mitigating the effects of
absent, partial inactivation, or abnormal expression of the Slo2 or Slo4
gene. The compositions are administered to a patient in an amount
sufficient to elicit a therapeutic response in the patient. An amount
adequate to accomplish this is defined as "therapeutically effective dose
or amount."

[0250]Delivery of the gene or genetic material into the cell is the first
step in gene therapy treatment of disease. A large number of delivery
methods are well known to those of skill in the art. Preferably, the
nucleic acids are administered for in vivo or ex vivo gene therapy uses.
Non-viral vector delivery systems include DNA plasmids, naked nucleic
acid, and nucleic acid complexed with a delivery vehicle such as a
liposome. Viral vector delivery systems include DNA and RNA viruses,
which have either episomal or integrated genomes after delivery to the
cell.

[0251]Methods of non-viral delivery of nucleic acids include lipofection,
microinjection, biolistics, virosomes, liposomes, immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, artificial
virions, and agent-enhanced uptake of DNA. Lipofection is described in,
e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No.
4,897,355 and lipofection reagents are sold commercially (e.g.,
Transfectam® and Lipofectin®). Cationic and neutral lipids that are
suitable for efficient receptor-recognition lipofection of
polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.
Delivery can be to cells (ex vivo administration) or target tissues (in
vivo administration).

[0253]The use of RNA or DNA viral based systems for the delivery of
nucleic acids take advantage of highly evolved processes for targeting a
virus to specific cells in the body and trafficking the viral payload to
the nucleus. Viral vectors can be administered directly to patients (in
vivo) or they can be used to treat cells in vitro and the modified cells
are administered to patients (ex vivo). Conventional viral based systems
for the delivery of nucleic acids could include retroviral, lentivirus,
adenoviral, adeno-associated and herpes simplex virus vectors for gene
transfer. Viral vectors are currently the most efficient and versatile
method of gene transfer in target cells and tissues. Integration in the
host genome is possible with the retrovirus, lentivirus, and
adeno-associated virus gene transfer methods, often resulting in long
term expression of the inserted transgene. Additionally, high
transduction efficiencies have been observed in many different cell types
and target tissues.

[0255]In applications where transient expression of the nucleic acid is
preferred, adenoviral based systems are typically used. Adenoviral based
vectors are capable of very high transduction efficiency in many cell
types and do not require cell division. With such vectors, high titer and
levels of expression have been obtained. This vector can be produced in
large quantities in a relatively simple system. Adeno-associated virus
("AAV") vectors are also used to transduce cells with target nucleic
acids, e.g., in the in vitro production of nucleic acids and peptides,
and for in vivo and ex vivo gene therapy procedures (see, e.g., West et
al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641;
Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.
94:1351 (1994)). Construction of recombinant AAV vectors are described in
a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et
al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell.
Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, Proc. Natl. Acad. Sci.
U.S.A. 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828
(1989).

[0256]Gene therapy vectors can be delivered in vivo by administration to
an individual patient, typically by systemic administration (e.g.,
intravenous, intraperitoneal, intramuscular, subdermal, or intracranial
infusion) or topical application, as described below. Alternatively,
vectors can be delivered to cells ex vivo, such as cells explanted from
an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue
biopsy) or universal donor hematopoietic stem cells, followed by
reimplantation of the cells into a patient, usually after selection for
cells which have incorporated the vector.

[0257]Ex vivo cell transfection for diagnostics, research, or for gene
therapy (e.g., via re-infusion of the transfected cells into the host
organism) is well known to those of skill in the art. In a preferred
embodiment, cells are isolated from the subject organism, transfected
with a nucleic acid (gene or cDNA), and re-infused back into the subject
organism (e.g., patient). Various cell types suitable for ex vivo
transfection are well known to those of skill in the art (see, e.g.,
Freshney et al., Culture of Animal Cells, A Manual of Basic Technique
(3rd ed. 1994)) and the references cited therein for a discussion of how
to isolate and culture cells from patients).

[0258]Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing therapeutic nucleic acids can be also administered directly to
the organism for transduction of cells in vivo. Alternatively, naked DNA
can be administered. Administration is by any of the routes normally used
for introducing a molecule into ultimate contact with blood or tissue
cells. Suitable methods of administering such nucleic acids are available
and well known to those of skill in the art, and, although more than one
route can be used to administer a particular composition, a particular
route can often provide a more immediate and more effective reaction than
another route.

[0259]Administration is by any of the routes normally used for introducing
a molecule into ultimate contact with blood or tissue cells. The nucleic
acids are administered in any suitable manner, preferably with
pharmaceutically acceptable carriers. Suitable methods of administering
such nucleic acids are available and well known to those of skill in the
art, and, although more than one route can be used to administer a
particular composition, a particular route can often provide a more
immediate and more effective reaction than another route.

IX. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

[0260]Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered (e.g., nucleic acid, protein,
modulatory compounds or transduced cell), as well as by the particular
method used to administer the composition. Accordingly, there are a wide
variety of suitable formulations of pharmaceutical compositions of the
present invention (see, e.g., Remington's Pharmaceutical Sciences,
17th ed., 1989). Administration can be in any convenient manner,
e.g., by injection, oral administration, inhalation, transdermal
application, or rectal administration.

[0261]Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the packaged nucleic
acid suspended in diluents, such as water, saline or PEG 400; (b)
capsules, sachets or tablets, each containing a predetermined amount of
the active ingredient, as liquids, solids, granules or gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet
forms can include one or more of lactose, sucrose, mannitol, sorbitol,
calcium phosphates, corn starch, potato starch, microcrystalline
cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,
stearic acid, and other excipients, colorants, fillers, binders,
diluents, buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers. Lozenge forms can comprise the active ingredient in a flavor,
e.g., sucrose, as well as pastilles comprising the active ingredient in
an inert base, such as gelatin and glycerin or sucrose and acacia
emulsions, gels, and the like containing, in addition to the active
ingredient, carriers known in the art.

[0262]The compound of choice, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be
"nebulized") to be administered via inhalation. Aerosol formulations can
be placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like.

[0263]Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the joints), intravenous, intramuscular,
intradermal, intraperitoneal, and subcutaneous routes, include aqueous
and non-aqueous, isotonic sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions that can include suspending
agents, solubilizers, thickening agents, stabilizers, and preservatives.
In the practice of this invention, compositions can be administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of administration.
The formulations of commends can be presented in unit-dose or multi-dose
sealed containers, such as ampules and vials.

[0264]Injection solutions and suspensions can be prepared from sterile
powders, granules, and tablets of the kind previously described. Cells
transduced by nucleic acids for ex vivo therapy can also be administered
intravenously or parenterally as described above.

[0265]The dose administered to a patient, in the context of the present
invention should be sufficient to effect a beneficial therapeutic
response in the patient over time. The dose will be determined by the
efficacy of the particular vector employed and the condition of the
patient, as well as the body weight or surface area of the patient to be
treated. The size of the dose also will be determined by the existence,
nature, and extent of any adverse side-effects that accompany the
administration of a particular vector, or transduced cell type in a
particular patient.

[0266]In determining the effective amount of the vector to be administered
in the treatment or prophylaxis of conditions owing to diminished or
aberrant expression of the Slo channels comprising a Slo2 or Slo4
subunit, the physician evaluates circulating plasma levels of the vector,
vector toxicities, progression of the disease, and the production of
anti-vector antibodies. In general, the dose equivalent of a naked
nucleic acid from a vector is from about 1 μg to 100 μg for a
typical 70 kilogram patient, and doses of vectors which include a
retroviral particle are calculated to yield an equivalent amount of
therapeutic nucleic acid.

[0267]For administration, compounds and transduced cells of the present
invention can be administered at a rate determined by the LD-50 of the
inhibitor, vector, or transduced cell type, and the side-effects of the
inhibitor, vector or cell type at various concentrations, as applied to
the mass and overall health of the patient. Administration can be
accomplished via single or divided doses.

X. KITS

[0268]Human Slo2 or Slo4 and their homologs are useful tools for examining
expression and regulation of potassium channels. Human Slo2 or
Slo4-specific reagents that specifically hybridize to hSlo2 or Slo4
nucleic acid, such as hSlo2 or Slo4 probes and primers, and hSlo2 or
Slo4-specific reagents that specifically bind to the hSlo2 or Slo4
protein, e.g., hSlo2 or Slo4 antibodies are used to examine expression
and regulation.

[0269]Nucleic acid assays for the presence of hSlo2 or Slo4 DNA and RNA in
a sample include numerous techniques are known to those skilled in the
art, such as Southern analysis, northern analysis, dot blots, RNase
protection, 51 analysis, amplification techniques such as PCR and LCR,
and in situ hybridization. In in situ hybridization, for example, the
target nucleic acid is liberated from its cellular surroundings in such
as to be available for hybridization within the cell while preserving the
cellular morphology for subsequent interpretation and analysis. The
following articles provide an overview of the art of in situ
hybridization: Singer et al., Biotechniques 4:230-250 (1986); Haase et
al., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic Acid
Hybridization: A Practical Approach (Hames et al., eds. 1987). In
addition, hSlo2 or Slo4 protein can be detected with the various
immunoassay techniques described above. The test sample is typically
compared to both a positive control (e.g., a sample expressing
recombinant Slo2 or Slo4 monomers) and a negative control.

[0270]The present invention also provides for kits for screening
modulators of the potassium channels of the invention. Such kits can be
prepared from readily available materials and reagents. For example, such
kits can comprise any one or more of the following materials: Slo2 or
Slo4 monomers, reaction tubes, and instructions for testing the
activities of potassium channels containing Slo2 or Slo4. A wide variety
of kits and components can be prepared according to the present
invention, depending upon the intended user of the kit and the particular
needs of the user. For example, the kit can be tailored for in vitro or
in vivo assays for measuring the activity of a potassium channel
comprising a Slo2 or Slo4 monomer.

[0271]All publications and patent applications cited in this specification
are herein incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to be
incorporated by reference.

[0272]Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to one of ordinary skill in
the art in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the spirit
or scope of the appended claims.

EXAMPLES

[0273]The following example is provided by way of illustration only and
not by way of limitation. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed or
modified to yield essentially similar results.

Example 1

Cloning and Expression of Human Slo2

[0274]A. Cloning

[0275]A fragment of the human Slo2 sequence was identified in the human
EST database at NCBI by BLAST search homology to the amino acid sequence
of human Slo1, a large-conductance calcium-activated potassium channel. A
single EST of Slo2, R87855, was identified, ordered and completely
sequenced. The clone contained approximately 700 bp of sequence with
homology to a long C-terminal cytoplasmic domain contained in the Slo1
sequence (Slo1 is over 3 Kb in length). Neither the 5' or 3' ends of the
gene were present in this clone, indicating that it represented only a
small fragment of the Slo2 gene. The cDNA was named Slo2 because it
shared the greatest homology with a C. elegans potassium channel that had
already been named Slo2. It should be noted, however, that human Slo2 and
the C. elegans gene are not orthologs.

[0276]The 3' end of the human Slo2 gene was cloned using two nested rounds
of RACE PCR with human hippocampus cDNA as a template. RACE PCR is a
technique in which a gene specific oligo is used in conjunction with an
oligo to a non-specific tag present on a pool of cDNAs to amplify the end
of the gene from that pool. In the first round, the gene-specific sense
primer used was 5'-CACCACGGAGCTCACCCACCCTTCC-3' (1) (SEQ ID NO:5). A
small aliquot of this reaction was then reamplified with a Slo2
gene-specific sense oligo nested 3' of (1),
5'-CGCGTCTTCAGCATCAGCATGTTGGAC-3' (2) (SEQ ID NO:6). Both gene specific
oligos were derived from the sequence of the R87855 EST clone. Four cDNAs
ranging from approximately 600 by to approximately 2 Kb were isolated and
sequenced. An approximately 1.8 Kb fragment was sequenced and found to
contain the 3' end of the Slo2 coding region as well as 3' UTR sequence.

[0277]Several rounds of 5' RACE PCR were used to clone most of the 5' end
of human Slo2. First, 2 rounds of nested 5' RACE PCR were used to amplify
an approximately 500 by fragment of Slo2 from a human hippocampus cDNA
library using Slo2-specific antisense oligos based on the R87855EST
sequence. In the first round, the gene specific antisense oligo used was
5'-CTGGTAGAGCAGTGTGTCCAACATGCTG-3' (3) (SEQ ID NO:7). A small aliquot of
this reaction was reamplified with a more 5'Slo2 antisense oligo
5'-ACTGCATGAAGCGCATGTTGGAAGGGTG-3' (4) (SEQ ID NO:8) to obtain the
fragment. This fragment contained significant homology to the C. elegans
Slo2 gene, but clearly did not represent a 5' complete cDNA.

[0278]Two new Slo2 antisense oligos were designed based on the 5' end of
this ˜500 by fragment.

[0279]2 rounds of nested 5' RACE PCR were used to obtain fragments of
˜1 Kb in length using these oligos. In the first round, the gene
specific antisense oligo used was 5'-CCCATTGCCGGCCGTCTCTGCCGAG-3' (5)
(SEQ ID NO:9). A small aliquot of this reaction was reamplified with a
more 5' Slo2 antisense oligo 5'-CTTGAACCCGTAGGCCTTGGCGTCTTC-3' (6) (SEQ
ID NO:10) to yield the 1 Kb fragments. These fragments encoded an amino
acid with over 30% identity to C. elegans Slo2, and with a lower level of
homology to Slo1. These fragments represented incomplete cDNAs because
the fragments are homologous to the middle of the C. elegans Slo2 gene,
which contains over 1 Kb of coding sequence upstream of the region
homologous to these human Slo2 fragments. The 5' PCR was repeated with 2
new human Slo2-specific antisense oligos based on the 5' most sequence
obtained from the above 5' RACE PCR fragments. The new oligos used were
5'-CACACCACGTGGTCAGCAAACTTGACG-3' (7) (SEQ ID NO:11) in the first round
and 5'-GCAGTTGGGGGCGAAGTCCTTCACGG-3' (8) (SEQ ID NO:12) in the second
round. A fragment of approximately 1.2 Kb was isolated from the second
reaction and sequenced. This band was found to contain homology to the 5'
region of C. elegans Slo2, but contained no obvious start codons.
Comparison to a newly cloned rat gene, rat SLACK (Joiner et al., Nat.
Neurosci. 1:462-9 (1998)), also indicated that the human Slo2 sequence
was probably 5' incomplete. The cDNA cloned by Joiner et al. appeared to
be the rat ortholog of human Slo2, sharing over 90% amino acid identity.

[0280]All RACE PCR products described above were produced under a
relatively standard set of conditions. Denaturations were carried out at
95° C. for 15 seconds, annealing temperatures ranged from
72-60° C. for 15 seconds, and extensions were carried out at
72° C. for 90 seconds to 3 minutes depending on the length of the
products. First round RACE reactions consisted of 35-40 cycles of PCR,
will nested reamplifications consisted of 20-30 cycles.

[0281]A clone containing a 5' incomplete (but otherwise full-length) human
Slo2 sequence was constructed using overlap extension PCR and 3 Slo2
fragments amplified from human hippocampus cDNA. A 5' fragment of
approximately 1.3 Kb was amplified using the sense oligo
5'-CACCTTCAAGGAGCGGCTCAAGCTG-3' (9) (SEQ ID NO:13) and the antisense
oligo 5'-GACGTGTGCACCAGCAGGGTGATGAG-3' (10) SEQ ID NO:14). The middle of
the Slo2 sequence was amplified as a 1.55 Kb fragment with the oligos
(sense) 5'-GTTTCACGTCAAGTTTGCTGACCACG-3' (11) (SEQ ID NO:15) and
(antisense) 5'-CCGTACGTGCGGATCCACAGGTCG-3' (12) (SEQ ID NO:16). The 3'
end of the Slo2 coding sequence was amplified with the sense oligo
5'-CGTGAAGGACTACATGATCACCATC-3' (13) (SEQ ID NO:17) and the antisense
oligo 5'-CAGGGTCTAGATTAGAGCTGTGTCTCGTCGCGAGTCTC-3' (14) (SEQ ID NO:18) to
produce a fragment of 800 bp. The latter oligo includes the predicted
Slo2 stop codon and 3' end of Slo2 coding (in bold), plus an XbaI site
for subcloning on the 5' end. It should be noted that only the bold
sequence corresponding to Slo2 is used to amplify Slo2. These fragments
were assembled into a single fragment using 2 rounds of standard overlap
extension PCR. First, the 5' and middle fragments were mixed and
amplified with oligos 9 and 12 to produce a fragment of approximately 2.8
Kb. Similarly, the middle and 3' end fragments were mixed and amplified
with oligos 11 and 14 to produce a fragment of approximately 2.3 Kb.
These two larger fragments were then mixed and amplified with oligos 9
and 14 to produce a fragment of 3.5 Kb containing all known human Slo2
sequence. This fragment was cloned into a plasmid vector and multiple
clones were sequenced to determine a final human Slo2 sequence for this
region. The conditions used to amplify the coding region of Slo2 were
similar to those described above for the Slo2 RACE reactions. One notable
exception is that longer extension times (4 minutes) were allowed during
the overlap reactions because of the large size of the desired fragments.

[0282]A potential 5' end for human Slo2 was identified by BLAST searching
the rat SLACK sequence against publicly available human genomic DNA
sequence. An exon homologous to the DNA sequence encoding amino acids
1-52 of rat Slack was identified. Based on homology to Rat SLACK, this
exon contained the 5' end of human Slo2 coding sequence. A complete
putative human Slo2 coding sequence was constructed using the sequence of
the 3.5 Kb construct described herein, the 5' end exon, and two other
exons identified in the BLAST search. BLAST analysis of this complete
coding sequence vs. the human Slo2 genomic sequence shows that the hSlo2
coding sequence is divided into at least 30 exons. This cDNA sequence
could not be predicted from genomic DNA, without the cloned cDNA.

[0283]The 5' end of Slo2 was amplified from human brain cDNA using an
overlap extension PCR screen. A fragment containing the start codon and
first 200 bp of Slo2 was amplified using the sense oligo
5'-CCACCATGGCGCGGGCCAAGCT-3' (15) (SEQ ID NO:19) and the antisense oligo
5'-GAGACAGGGAGGAGTCCAGGCTGAA-3' (16) (SEQ ID NO:20). Only the bold bases
in 15 match Slo2 and are used for amplification of Slo2; the 5' bases add
a Kozak consensus sequence and are included only for expression vector
construction. A second fragment of approximately 400 by that overlapped
the first fragment and a unique Hind III restriction site in the 3.5 Kb
Slo2 clone was amplified using the oligos 5'-CGTGGGCCAGAGGCTTCCTGTAGAA-3'
(17) (SEQ ID NO:21) and 5'-GCTCCCAGATGTTGCCTTTGTAGCTG-3' (18) (SEQ ID
NO:22). These two fragments were mixed and amplified with oligos 15 and
18 to produce an approximately 550 by fragment containing both the
initiator methionine of Slo2 and the unique Slo2 Hind III site. This
fragment was cloned into a standard plasmid and 3 clones were sequenced.
Each clone was identical to the consensus human Slo2 derived from our
cDNA and genomic information. A full length Slo2 coding region was then
assembled by joining the 5' end fragment and 3.5 Kb Slo2 fragment at
their common Hind III restriction site by standard DNA cloning methods.

[0284]Recently, a large but partial human Slo2 cDNA was deposited in the
NCBI database (KIA1422, accession number AB037843). This clone differs
from the complete human Slo2 clone described above in several key ways
(see FIG. 1). Most importantly, it is incomplete on the 3' end. DNA
encoding 127 amino acids at the carboxy terminus of human Slo2 is missing
from KIAA1422. Additionally, KIAA1422 has an alternative 5' end. Because
the KIAA1422 reading frame remains open at the 5' end, it is unclear
whether the KIAA1422 cDNA represents a clone with a complete alternative
5' end (with the protein starting at one of the internal methionine
codons), or whether it represents a 5' incomplete clone. Finally,
KIAA1422 contains a 2 amino acid insertion (GT) at the equivalent
position of amino acid 650 in the hSlo2 sequence.

[0285]The numbered oligonucleotides listed above can be used in various
combinations to amplify sections of the hSlo2 cDNA from an appropriate
template, such as human brain cDNA, using the conditions described above.
Oligos 14-17 are not contained in the KIAA1422 sequence and oligo pairs
including at least one of these oligos can be used to amplify fragments
that could not be derived from the KIAA1422 sequence. Oligo 17 can be
paired with 18, 8, 7, 6, 5, 4, 3 and 14 to produce fragments of
approximately 415 bp, 1.25 Kb, 1.3 Kb, 2.19 Kb, 2.23 Kb, 2.59 Kb, 2.74 Kb
and 3.58 Kb, respectively. Oligo 15 can be substituted for 17 in the
above combinations to produce fragments that are approximately 150 by
longer than those listed above. Additionally, oligo 14 can be paired with
9, 11, 1, 2 and 13 to produce fragments of approximately 3.49 Kb, 2.29
Kb, 1.03 Kb, 880 by and 830 bp, respectively. None of the oligo pairs
listed above will amplify KIAA1422. If at least one of these
amplifications can be obtained from a gene, and the sequence of the
fragment is substantially identical to that of human Slo2, then the
sequence should be considered a species of human Slo2. Other notable
pairs that would amplify both KIAA1422 and human Slo2 are 9+10 and 11+12,
which were used in the production of the 3.5 Kb 5' incomplete human Slo2
clone.

[0286]B. mRNA Expression

[0287]A northern blot and mRNA dot blot probed with a 32P-labeled PCR
fragment produced with oligos 13 and 14 are shown in FIG. 3. On the
northern blot, prominent band of approximately 5 Kb is seen in brain,
with a less intense band visible at roughly 6 Kb. Similar bands are seen
in skeletal muscle, albeit at a lower intensity. Faint signals are seen
in heart and spleen. The mRNA dot blot shows widespread expression of
human Slo2 in the central nervous system. Expression is highest in the
cerebellum, cerebral cortex, occipital lobe, temporal lobe, putamen and
nucleus accumbens. Significant levels of expression were also found in
the fetal brain, amygdala, caudate nucleus, frontal lobe, hippocampus,
substantia nigra and thalamus. In addition to the peripheral tissues
identified as expressing Slo2 on the northern blot, expression
significant levels of expression were found in ovary, placenta, and fetal
spleen.

[0288]C. Xenopus Expression

[0289]Functional expression of human Slo2 was examined in Xenopus oocytes.
Slo2 was cloned into the pOX expression vector and run-off cRNA
transcripts were prepared. These transcripts were injected in mature
stage 4 Xenopus oocytes and examined under whole cell two-electrode
voltage clamp after 24-48 hours. Oocyte expression procedures were
performed according to Jegla & Salkoff, J. Neurosci 17(1):32-44 (1996)).

[0290]FIG. 5A shows a series of currents recorded from an oocyte
expressing human Slo2. A large, outwardly rectifying current is seen in
voltage steps above -100 mV. No similar current was seen in uninjected
control oocytes. The reversal potential of the human Slo2 current shifted
with changes in external potassium concentration (FIG. 5B). In the
example shown, the reversal potential of the Slo2 current shifts almost
+70 mV in response to an increase in external potassium concentration
from 2 mM to 50 mM. This large shift is almost as much as that predicted
for a channel that is perfectly potassium selective, and indicates that
Slo2 channels are highly selective for potassium over other cations.
These results also indicate that Slo2 is voltage gated.

Example 2

Cloning and Expression of Slo4

[0291]A. Cloning

[0292]Partial human Slo4 sequences were originally identified with TBLASTN
searches of 3 databases with the rat SLACK sequence and partial human
Slo2 sequences: A proprietary database, the public EST database at NCBI,
and the public Genome Survey Sequence Database at NCBI. The proprietary
clone 5035170 contained a short stretch of Slo4 coding sequence with
amino acid 60% identity to rat SLACK amino acids 646-730. The entire
clone had an insert of less than 700 bp. It was sequenced in its entirety
and determined that most of the insert probably represented intronic
sequence. The two Genome survey sequences (GSS), AQ701228 and AQ892600
contained homology Rat SLACK just 5' and 3' to the proprietary clone,
respectively. Finally, a public EST clone (AI791929) was identified that
had homology to the 3' end of the RAT SLACK coding sequence and appeared
to contain a stop codon for the Slo4 open reading frame. These
non-overlapping sequences were confirmed to have come from the same gene
by amplifying an approximately 1.5 Kb fragment with a sense oligo based
on the 5' most sequence (AQ701228), 5'-GGCGTCTGCTTGATTGGTGTTAGGA-3' (19)
(SEQ ID NO:23), and an antisense oligo overlapping the stop codon in the
EST sequence, 5'-TTTATCTAGAATCAAAGTTGAGTTTCCTCCCGAG-3' (20) (SEQ ID
NO:24). This amplified clone contained sequence identical to all four of
the clones identified by BLAST searches. It also contained a high degree
of homology to human Slo2 and rat SLACK across its entire length.

[0293]Two partial genomic sequences of Slo4 (AL139137.1 and AL138931.1)
were then discovered using TBLASTN searches of the NCBI High Throughput
Genomic Sequence Database (HTGS) with the complete human Slo2 sequence
described above. AL138931.1 contained 10 exons with homology to Slo2,
with 3 of those exons containing sequence more 5' than that which we had
previously identified. The 5' most exon contained homology to amino acids
449-484 of the human Slo2 sequence. To clone the 5' end of the Slo4
coding sequence, antisense oligos were designed based on this new
sequence for use in 5' RACE PCR. A single round of RACE PCR with the
Slo4-specific antisense oligo 5'-CCCGGAGCATCTACCGTACATCTTC-3' (21) (SEQ
ID NO:25) produced a fragment of approximately 800 by from human brain
cDNA. This fragment extended the Slo4 coding sequence by almost 500 by
into a region highly homologous to the pore-loop motifs of Slo potassium
channels.

[0294]The 5' end of the Slo4 coding sequence was cloned with 2 nested
rounds of 5' RACE PCR using Slo4-specific antisense oligos based on the
new sequence obtained in the first 5' RACE attempt. The Slo4-specific
oligos used were 5'-CCAGCTGTTCAAACTGTATGGGTAG-3' (22) (SEQ ID NO:26) and
5'-GCTTGGAGGACCATGTTTCAGGAGT-3' (23) (SEQ ID NO:27) in the first and
second rounds, respectively. Conditions for these amplifications and the
first Slo4 5' RACE attempt were similar to those described above for
Slo2. An approximately 900 by fragment was isolated from the 2nd reaction
and was determined to contain the 5' end of the Slo4 coding sequence. The
fragment contains a long open reading frame with substantial homology to
the 5' coding region of human Slo2. A stop codon ends the open reading
frame immediately upstream of a methionine codon, indicating that this is
the initiator methionine of Slo4.

[0295]The entire Slo4 coding sequence was amplified in a single fragment
using a sense oligo overlapping the initiator methionine codon,
5'-ATCCCAATTGCCGCCATGGTTGATTTGGAGAGCGAAGTG-3' (24) (SEQ ID NO:28) and the
antisense oligo overlapping the stop codon, oligo #20. Only the bases
listed in bold type in oligos 20 and 24 match the Slo4 DNA sequence. In
oligo 20, the additional bases at the 5' end add an XbaI restriction site
to assist subcloning. In oligo 24 the additional bases at the 5' end add
a MunI site for subcloning and a Kozak consensus sequence to boost
translation initiation at the Slo4 methionine codon. Only the areas given
in bold type are used for the amplification of Slo4; the other bases need
not be present to obtain amplification.

[0296]Two additional Slo4-specific sense oligos and one Slo4-specific
antisense oligo can be used to amplify Slo4:

[0297]These oligos can be used in combination with the other Slo4 oligos
listed above to amplify a variety of Slo4 fragments from an appropriate
cDNA source such as human brain. 24 can be used with 23, 22, 21, 27 or 20
to produce fragments of approximately 780 bp, 830 bp, 1.46 Kb, 1.98 Kb
and 3.4 Kb, respectively. 25 can be used with 21, 27 or 20 to produce
bands of around 250 bp, 780 by and 2.2 Kb, respectively. 19 can be used
with 27 or 20 to produce fragments of approximately 420 by and 1.85 Kb.
Finally, 26 can be used with 20 to produce a fragment of around 600 bp.
If at least one of these amplifications can be obtained from a gene, and
the sequence of the fragment is substantially identical to that of human
Slo4, then the sequence should be considered a species of human Slo4.

[0298]B. mRNA Expression

[0299]A human northern blot and dot blot hybridized with a
32P-labeled Slo4 cDNA probe are shown in FIG. 7. A transcript of
approximately 5.5 Kb is labeled in most of the tissues on the northern
blot. Expression is highest in the liver, with high level expression also
being found in the brain and heart. Lower levels of expression are
detected in skeletal muscle, colon, spleen, kidney, small intestine,
placenta and lung. Larger transcripts of approximately 9 Kb and 13 Kb are
seen in brain and heart. These may represent alternative transcripts or
incompletely processed transcripts. A 4.5 Kb transcript is seen in lung,
and may represent an alternative transcript; it is long enough to encode
a complete Slo4 protein. In the dot blot, the highest levels of
expression are seen in liver, fetal brain, fetal kidney and fetal liver.
High levels of expression are also seen in testis, fetal lung, most brain
regions, the atrium, the GI track, lung, placenta and bladder. Expression
is detectable in many other tissues on the blot. Based on comparison with
the northern results in (A), these low signals are likely to represent
Slo4 expression and not background non-specific hybridization.

[0300]C. Xenopus Expression

[0301]Functional expression of human Slo4 was examined in Xenopus oocytes.
Slo4 was cloned into the pOX expression vector and run-off cRNA
transcripts were prepared. These transcripts were injected in mature
stage 4 Xenopus oocytes and examined under whole cell two-electrode
voltage clamp after 24-48 hours. Oocyte expression procedures were
performed according to Jegla & Salkoff, J. Neurosci 17(1):32-44 (1996)).

[0302]FIG. 6 shows a similar set of experiments conducted with human Slo4.
As observed for Slo2, large, outwardly rectifying potassium currents are
seen with depolarizing voltage steps. The reversal potential for Slo4 was
also highly sensitive to changes in external potassium concentration. As
shown in FIG. 6B, change in external potassium concentration from 2 mM to
20 mM caused the Slo4 current reversal potential to shift over +45 mV.
This change is almost at great at the +48 mV shift that would be expected
for a perfectly potassium selective channel, indicating that Slo4 is very
highly potassium selective over other cations, as is Slo2. These results
also indicate that Slo4 is voltage gated.